# The Changes in Starch Gelatinization Behavior under the Influence of Acetic Acid in Vegetable Sponge Cake Batter in Order to Obtain New Snacks

Manon Chemin, Olivier Paurd, Laure Villaceque, Alain Riaublanc, Patricia Le-Bail

► To cite this version:

Manon Chemin, Olivier Paurd, Laure Villaceque, Alain Riaublanc, Patricia Le-Bail. The Changes in Starch Gelatinization Behavior under the Influence of Acetic Acid in Vegetable Sponge Cake Batter in Order to Obtain New Snacks. Polymers, 2022, 14 (19), pp.4053. 10.3390/polym14194053. hal-04131375

HAL Id: hal-04131375

https://hal.inrae.fr/hal-04131375

Submitted on 16 Jun 2023

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
Article

# The Changes in Starch Gelatinization Behavior under the Influence of Acetic Acid in Vegetable Sponge Cake Batter in Order to Obtain New Snacks

Manon Chemin<fnref n="1" />,<fnref n="2" />, Olivier Paurd<fnref n="2" />, Laure Villaceque<fnref n="2" />, Alain Riaublanc<fnref n="1" /> and Patricia Le-Bail<fnref n="1" />,*

**Abstract:** (1) Background: Adding white vinegar to the batter of a sponge cake without biological fermentation requires the effects of acidification on the batter to be checked, in particular concerning batter-to-crumb transition. (2) Methods: µDSC analyses were carried out on three batters formulated from flour, colza oil, salt, carrot, and water with or without the addition of white vinegar. (3) Results: Wheat, chickpea, and quinoa starches had gelatinization temperatures ($T_{Ge}$) of 60.1, 72.4, and 70.5 °C at batter humidity and gelatinization enthalpies ($\Delta H_{Ge}$) of 9.2, 15, and 9.1 J/g$_{dry}$ starch. Due to the effect of the salt and carrot, the corresponding wholemeal batter had $T_{Ge}$ of 64.2, 74.1, and 72.4 °C and $\Delta H_{Ge}$ of 10.5, 15.3, and 10.9 J/g$_{dry}$ starch. Acidified batters at pH 4 saw their $T_{Ge}$ decrease, and their enthalpies increase compared to the controls. The calorimetric study of model mixtures revealed three different evolutions of $\Delta H_{Ge}$ as a function of pH, explained by the isoelectric behavior of flours and/or the attack of starch by acetic acid. (4) Conclusions: These results could be useful for adapting the cooking step of the acid batter in order to produce alternative snacks.

**Keywords:** vegetable sponge cake; wheat flour; chickpea flour; quinoa flour; acetic acid; micro-differential scanning calorimetry

<figure><img src="image_2.png" /></figure>

check for
updates

**Citation:** Chemin, M.; Paurd, O.; Villaceque, L.; Riaublanc, A.; Le-Bail, P. The Changes in Starch Gelatinization Behavior under the Influence of Acetic Acid in Vegetable Sponge Cake Batter in Order to Obtain New Snacks. *Polymers* **2022**, *14*, 4053. https://doi.org/10.3390/polym14194053

Academic Editors: Dimitrios Bikiaris and Amit Kumar

Received: 13 July 2022
Accepted: 22 September 2022
Published: 27 September 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

<figure><img src="image_3.png" /></figure>

**Copyright:** © 2022 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

## 1. Introduction

Almost half (47%) of French people have an aperitif at least once a week [1]. With around 250 varieties of crunchable appetizers including crisps, tuiles, salted seeds, extrudates and biscuits, the appetizer snack sector is expected to grow by 4.5% between 2019 and 2024 globally [2]. The aperitif snack market has been evolving, integrating new production techniques, new flavors and bringing new societal, environmental, and nutritional dimensions to the products. In particular, the offer is adapting to new ways of consuming, by offering local, organic, gluten-free and up-cycling in the formulations [3]. Traditionally, crackers are made using a multi-step process: (1) fermentation of a dough made from wheat flour, (2) rolling the batter and cutting the pieces, (3) cooking [4]. Several effects of fermentation have been noted [5]: during this stage, the rheology of the dough evolves, allowing its lamination [6], aromatic molecules are formed, as well as acid molecules causing the decrease of pH.

In the context of our study, the use of an alternative process for manufacturing snacks, obtained by drying a sponge cake [7], does not involve a biological fermentation step. In order to compensate for this absence, the addition of white vinegar was considered. Indeed, beyond the sensory interest, the presence of vinegar in food formulations is interesting at the functional level. First, the acidic pHs caused by vinegar allow better preservation of products through their antibacterial effect [8–10] and can reach optimal values to reinforce the action of other raw materials, such as the activity of flour proteolytic enzymes [5], the biocidal effect of preservatives [11] and the antioxidant effect of certain ingredients [12].

<footnote n="1">¹ INRAE, UR1268 Biopolymers Interactions Assemblies, Impasse Thérèse Bertrand-Fontaine, BP 71627, CEDEX 3, 44316 Nantes, France</footnote>

<footnote n="2">² Nbread Process, 3 Boulevard de l'industrie, 41700 Contres, France</footnote>

<footnote>* Correspondence: patricia.le-bail@nantes.inrae.fr</footnote>
The acidity induced by vinegar can also limit the formation of carcinogenic compounds, i.e., acrylamides, sometimes produced during the cooking of snacks [13]. Next, vinegar has certain health benefits, through its antibacterial and antioxidant activity [14]. Finally, the acetic acid of the vinegar can bring new properties to the mixtures by interacting with the other ingredients of the recipe. Indeed, many studies have been carried out on the use of different acids for the treatment of starch.

To our knowledge, acetic acid has been studied on wheat, potato, rice, and tapioca starch [15–17]. We want to provide new knowledge on the treatment of chickpea and quinoa flour with acetic acid. In the literature, there are also works relating to the study of the gelatinization of wheat, chickpea, and quinoa starches in the presence of HCL and H₂SO₄, but under lintnerization conditions [18–20]. Other studies, although fewer in number, have focused on the acidification of real batters but described only analysis of the acidification action on the finished product, for a formula of crackers with buckwheat [21], or formula of gluten-free bread [22].

One of the primary objectives of this invention is to produce high-fiber snacks using so-called "ugly" vegetables deemed unsuitable for sale, the batter studied contains a high content of vegetable fibers. For these snacks made using an innovative process, it is important to know the energy needed to allow complete gelatinization of the starch system in the batter during cooking. The objective of this work is therefore to study and compare the changes in gelatinization behavior under the influence of acetic acid of different starches (wheat, chickpea, and quinoa), present in acidified vegetable sponge cakes.

## 2. Materials and Methods

### 2.1. Materials

Organic T45 wheat flour (Fw) (moisture 16.6%, starch 68.5% including 31.2% amylose and 68.8% amylopectin, protein 12%, fat 0.8%) was supplied by the Giraudineau mill (Saint Colomban, France). Desi variety organic chickpea flour (FCP (moisture 10.9%, starch 40.9% including 32.4% amylose and 67.6% amylopectin, protein 16.8%, fat 6.3%) was provided by Qualisol (Castelsarrasin, France). Quinoa flour (FQ) (moisture 9.9%, starch 58.4% including 18.8% amylose and 81.2% amylopectin, protein 11.4%, fat) was obtained from seeds of Blond Berry Quinoa purchased from SAS Berry Graines (Nérondes, France). The unshelled seeds were crushed with a Retsch ZM200 brand ultracentrifugal grinder (Retsch, Haan, Germany) consisting of a 12-tooth rotor and a 0.2 mm mesh sieve. The carrot puree (C) (moisture 92.7%) was provided by Clément Faugier ETS (Privas, France) in frozen form. The puree pebbles were put in a cold room at 4 °C for 48 h before being used to defrost them. Rapeseed oil (O) (7.6% saturated fatty acids, 63.1% monounsaturated fatty acids, and 29.3% polyunsaturated fatty acids), fine table salt (Sa), and white vinegar (acetic acid, acidity 7.2%, pH 2.8) were bought in supermarkets (Nantes, France). The water (W) was millipore water, bubbled under N₂ before use to overcome oxidation phenomena.

### 2.2. Sample Preparation

#### 2.2.1. Preparation of the Vegetable Sponge Cake Batter

The reference industrial sponge cake batter recipe had the particularity of being egg-free, gluten-free, yeast-free, and additive-free. The various reference recipes contain 30% of flour, 5% of oil, 0.3% of salt, 50% of carrot and 14.7% of added water. The formulations are written in dry basis as follows:

$$F_a O_b Sa_c C_d W_y$$

F: dry flour, O: rapeseed oil, Sa: salt, C: dry carrot, W: total water present in the mixture a/b; a/c; and a/d: constant

$$a + b + c + d + y = 100$$

The different formulations are described in Table 1.
<table><caption>**Table 1.** Formulations of the different sponge cake batters with and without carrots.</caption><thead><tr><th>Formulation</th><th>Dry Flour (g)</th><th>Oil (g)</th><th>Salt (g)</th><th>Dry Carrot (g)</th><th>Water (g)</th></tr></thead><tbody><tr><td>F<sub>w29</sub> O<sub>5.6</sub> Sa<sub>0.4</sub> W<sub>65</sub></td><td>29</td><td>5.6</td><td>0.4</td><td>0</td><td>65</td></tr><tr><td>F<sub>CP29</sub> O<sub>5.6</sub> Sa<sub>0.4</sub> W<sub>65</sub></td><td>29</td><td>5.6</td><td>0.4</td><td>0</td><td>65</td></tr><tr><td>F<sub>Q29</sub> O<sub>5.6</sub> Sa<sub>0.4</sub> W<sub>65</sub></td><td>29</td><td>5.6</td><td>0.4</td><td>0</td><td>65</td></tr><tr><td>F<sub>w26</sub> O<sub>5</sub> Sa<sub>0.4</sub> C<sub>3.6</sub> W<sub>65</sub></td><td>26</td><td>5</td><td>0.4</td><td>3.6</td><td>65</td></tr><tr><td>F<sub>CP26</sub> O<sub>5</sub> Sa<sub>0.4</sub> C<sub>3.6</sub> W<sub>65</sub></td><td>26</td><td>5</td><td>0.4</td><td>3.6</td><td>65</td></tr><tr><td>F<sub>Q26</sub> O<sub>5</sub> Sa<sub>0.4</sub> C<sub>3.6</sub> W<sub>65</sub></td><td>26</td><td>5</td><td>0.4</td><td>3.6</td><td>65</td></tr></tbody></table>

All the ingredients, balanced at room temperature, were weighed one by one then mixed in a cylindrical stainless-steel tank with a capacity of 2.5 L, using a mixing blade. The mixing speed was set at 150 revolutions per minute. The mixing time was 15 min for all batters.

### 2.2.2. Preparation of Reference Model Mixtures

The reference model mixtures were flour and water ($F_xW_y$) primary model media. These simplified mixtures were made for two humidity values: the reference value (65%) and excess water (80%).

### 2.2.3. Acidification with White Vinegar

New sponge cake batters were formulated at a target pH of 4 by replacing part of the water in the formulas with white vinegar (Table 2).

<table><caption>**Table 2.** Formulations of the different acidified sponge cake batters with and without carrots.</caption><thead><tr><th>Formulation</th><th>Dry Flour (g)</th><th>Oil (g)</th><th>Salt (g)</th><th>Dry Carrot (g)</th><th>Water (g)</th><th>White Vinegar (g)</th></tr></thead><tbody><tr><td>F<sub>w29</sub> O<sub>5.6</sub> Sa<sub>0.4</sub> P<sub>65</sub></td><td>29</td><td>5.6</td><td>0.4</td><td>0</td><td>60.3</td><td>4.7</td></tr><tr><td>F<sub>CP29</sub> O<sub>5.6</sub> Sa<sub>0.4</sub> P<sub>65</sub></td><td>29</td><td>5.6</td><td>0.3</td><td>0</td><td>36.3</td><td>28.6</td></tr><tr><td>F<sub>Q29</sub> O<sub>5.6</sub> Sa<sub>0.4</sub> P<sub>65</sub></td><td>29</td><td>5.6</td><td>0.4</td><td>0</td><td>42.8</td><td>22.2</td></tr><tr><td>F<sub>w26</sub> O<sub>5</sub> Sa<sub>0.4</sub>C<sub>3.6</sub> P<sub>65</sub></td><td>26</td><td>5</td><td>0.4</td><td>3.6</td><td>54.1</td><td>10.9</td></tr><tr><td>F<sub>CP26</sub> O<sub>5</sub> Sa<sub>0.4</sub> C<sub>3.6</sub>P<sub>65</sub></td><td>26</td><td>5</td><td>0.4</td><td>3.6</td><td>45.5</td><td>19.5</td></tr><tr><td>F<sub>Q26</sub> O<sub>5</sub> Sa<sub>0.4</sub>C<sub>3.6</sub> P<sub>65</sub></td><td>26</td><td>5</td><td>0.4</td><td>3.6</td><td>45.5</td><td>19.5</td></tr></tbody></table>

To simplify the coding in results and discussion parts, the proportion of each component in the mixture will no longer be specified.

### 2.3. pH Measurement

The pH of the batter was determined using a calibrated pH meter with a probe for pastes (Hanna instruments) at room temperature.

### 2.4. Zeta Potential of Flours

For each flour, a range of mixtures have been made in the presence of vinegar and water (55 mg of flour for 100 g of plasticizer) to reach 4 different pH values from 3 to 6. For each mixture, zeta potential has been measured by electrophoretic light scattering with a Zetasizer analyser (Malvern Panalytical, Malvern, UK). After equilibration during 20 min at 20 °C. The operation has been made on 5 samples per mixture

### 2.5. Micro Differential Scanning Calorimetry (μDSC)

The gelatinization properties of the starches were studied for all the mixtures with a μDSC 7 microcalorimeter (Setaram, Caluire, France). An empty cell was used as a
reference. 300 to 500 mg of the sample were placed in a sealed µDSC Hastelloy capsule for thermal analysis. After equilibration at a temperature of 10 °C and maintaining at this temperature for 10 min, a rise from 10 to 120 °C at a rate of 1 °C/minute was carried out. The thermograms obtained were normalized in J/g of dry starch for analysis. The gelatinization peak temperature ($T_{Ge}$) and the gelatinization enthalpy ($\Delta H_{Ge}$) were determined. In addition, the partial melting enthalpy was calculated from the start of the endotherm (in 1 °C increments) to plot the curve representing the cumulative enthalpy values as a function of temperature.

## 2.6. Statistical Analysis

For each formulation, 3 batters were produced, and 3 batter samples were taken per production. The mean and standard deviation were calculated, and a one-way ANOVA analysis was performed with Stat graphics software.

# 3. Results and Discussion

## 3.1. The pH of the Different Mixtures

Carrots benefit from a pH close to 6.0. This pH is relatively high compared to fruits such as orange or apple, which have pH values below 4.5, which eliminated an added effect not related to acidification. Table 3 brings together the pH values of the different mixtures.

<table><caption>**Table 3.** pH of the different sponge cake batters.</caption><thead><tr><td>Mixtures</td><td>pH</td></tr></thead><tbody><tr><td>F<sub>w</sub>OSaW</td><td>5.97 +/− 0.01</td></tr><tr><td>F<sub>w</sub>OSaCW</td><td>5.89 +/− 0.06</td></tr><tr><td>F<sub>CP</sub>OSaW</td><td>6.04 +/− 0.03</td></tr><tr><td>F<sub>CP</sub>OSaCW</td><td>5.83 +/− 0.09</td></tr><tr><td>F<sub>Q</sub>OSaW</td><td>6.36 +/− 0.02</td></tr><tr><td>F<sub>Q</sub>OSaCW</td><td>6.16 +/− 0.02</td></tr></tbody></table>

## 3.2. Study of the Gelatinization of the Three Flours and Batters FOSaW and FOSaCW

Three different flours were used for this study. Wheat, a cereal flour traditionally used in bakery/pastry products and chickpea and quinoa, two alternative flours, one legume, the other pseudocereal, which have several industrial interests: on the one hand a nutritional aspect (gluten-free flours, but rich in proteins), on the other hand interesting features (emulsifying and foaming properties allowing them to replace egg white). Indeed, they had already given satisfactory results on dough rheology, bread texture and sensory analysis [23–25].

Wheat flour with excess water (80%) showed a gelatinization temperature $T_{Ge}$ equal to 59.6 +/− 0.2 °C which was close to the results of Sopade et al. (60.3 °C) [26]. The measured enthalpy $\Delta H_{Ge}$ was 9.7 +/− 0.4 J/g<sub>dry starch</sub> (9.2 +/− 0.2 J/g<sub>dry starch</sub> for Sopade).

For chickpea flour, the gelatinization temperature and enthalpy found were $T_{Ge}$ = 70.4 °C +/− 0.4 °C and $\Delta H_{Ge}$ = 12.6 J/g<sub>dry starch</sub>. In the literature, the authors mentioned slightly higher $T_{Ge}$ gelatinization temperatures (70.6 to 73.3 °C) and much lower enthalpies (<5 J/g flour) [27–29]. However, the difference was explained by the fact that these authors did not relate the enthalpy to the quantity of dry starch. Quinoa flour had a gelatinization temperature $T_{Ge}$ of 68.2 +/− 0.2 °C and a gelatinization enthalpy of 8.4 +/− 0.5 J/g<sub>dry starch</sub>. These results were different from those mentioned in the literature [30,31], with lower gelatinization temperatures (64.5 °C and 67 °C, respectively) and generally higher enthalpies (16.8 J/g and 14.6 J/g, respectively). The differences observed between all the studies can be explained by the variety of composition (botanical origin, amount of amylose, amylopectin, starch in flour, and other constituents), differences in structure (organization of amylose and of amylopectin) [32], and the treatment of the flours used (cultivation conditions, milling, extraction, storage).
The T<sub>Ge</sub> gelatinization temperatures of chickpea and quinoa flour were shifted to higher temperatures 72.4 +/− 0.1 °C and 70.5 +/− 0.1 °C, respectively, whereas they were stable for wheat starch when the water content decreased (65%). A more or less marked increase in T<sub>Ge</sub> when the water content decreases has been observed for mixtures with chickpea flour [27] and also buckwheat [33], chestnut [34], and rice flour [35]. Moreover, as for wheat flour, a shoulder appeared on the Ge endotherm, which turned into a starch melting endotherm, M1 moving towards higher temperatures with decreasing water content. On the other hand, the enthalpies of gelatinization were not significantly different.

Batters FOSaW where F represented the flour (wheat or chickpea or quinoa), O the fat, S the salt, and W the total water of the system, had a higher gelatinization temperature (Figure 1).

<figure><img src="image_4.png" /><figcaption>**Figure 1.** Gelatinization temperatures and enthalpies of different batters (with and without carrot and vinegar).</figcaption></figure>

This slight increase in the gelatinization temperature was linked to the presence of salt which competed with the starch for access to water. This competitive hydration resulted in less availability of water for starch gelatinization. D'Appolonia in 1972 and Spies and Hoseney in 1982 attributed the increase in gelatinization temperature to the solute's ability to limit the amount of water available to starch, i.e., to reduce the water chemistry potential [36,37]. However, according to Wootton, the effect of salt on starch gelatinization was not solely due to the decrease in water availability since the energy of
gelatinization did not decrease at the same rate as the amount of salt added to the aqueous phase [38]. The enthalpies of the FOSaW batters were not significantly modified compared to those obtained for F₃₅W₆₅ primary systems. This can be explained by the fact that the salt concentrations in our systems were very low (1.5 to 2% of the dry weight of the starch), which reduced its effect. Chiotelli and al [39] observed a decrease in the gelatinization enthalpy for wheat starch in presence of 16% of salt whereas there was no effect with low concentration of salt.

For the 3 complete batters FOSaCW where C represented the carrot, the differential enthalpy analysis study (Figure 1) showed that the addition of carrots to the batter led to a slight increase in temperature and in the gelatinization enthalpy for the three flours. For these systems, the majority of the water was provided by the carrot. The fiber content of the carrot was 2.7 g/100 g, the water provided by the carrot was therefore partly bound in the fibers and perhaps less accessible by the starch. However, at 65% water, the systems studied were still considered very close to excess water. The fibers, at this water content, therefore, did not cause any significant hindrance to gelatinization.

In the literature, fibers are added in the form of soluble or insoluble fibers. It was shown that insoluble or partially soluble fiber provided major effects on gelatinization (TGe decrease and ΔH increase). For example, Santos et al. [40] have studied the effects of flour replacement at different levels (6–34%) by soluble (inuline), partially soluble (sugar beet, pea cell wall), and insoluble (pea hull) fibers. Thermal profiles of wheat dough containing different fibers have been investigated by simulating baking, cooling, and storage in differential scanning calorimetry (DSC) pans. In general, dietary fiber incorporation into water-flour systems delayed endothermic transition temperatures for both gelatinization and retrogradation phenomena. Z. Goranova, et al. [41] evaluated the influence of functional ingredients on starch gelatinization in the sponge cake batter. They reduced the quantity of wheat flour in the sponge cake batter and added functional ingredients of 50% einkorn wholemeal flour, or 20% Jerusalem artichoke powder, or 35% cocoa husk powder. They showed that gelatinization occurs at higher temperatures and with higher energy consumption. They concluded that the retarding effect of the functional ingredients is related to the water binding capacity and the presence of dietary fiber.

We agreed with the literature but the water content of our sponge cakes being higher, the effect was less visible and for an equivalent water content our fiber content was lower.

### 3.3. *Impact of Acetic Acid on the Six Batters FOSaW and FOSaCP*

The study also focused on the impact of the acidification of a vegetable sponge cake batter with white vinegar on the thermal properties of the mixture. In order to determine the quantity of acetic acid to be used in the mixtures to reach a targeted pH of 4, a pH determination analysis was carried out on the batter with or without the presence of carrots.

A differential enthalpy analysis study was then conducted on FOSaCP batter to observe the hydrothermal properties of acidified batter. Gelatinization temperatures and enthalpies were shown in orange in Figure 1. A decrease in gelatinization temperature was observed when acetic acid was added and was much less marked for wheat than for chickpea and quinoa batters. Conversely, the gelatinization enthalpy increased for the three flours. Majzoobi and Beparva [15] observed the same effect on wheat starch treated with acetic acid for the T<sub>Ge</sub> but the opposite effect for the gelatinization enthalpy and they suggested that the crystalline structure of the starch molecules inside the granules, was affected by the acids and hence less energy required to melt the crystalline structure. Likewise, Ohishi and collaborators [16] showed by DSC measurements that rice starch gelatinized more easily in the presence of acetic acid, they found that the addition of 0.2 M acetic acid resulted in a significantly lower gelatinization temperature (~1 °C) for rice starch. Hibi [42] also reported that the endothermic peak temperature of rice starch was lower in the presence of 0.33 M acetic acid. However, in both previous cases, the resultant pH of the heated starch dispersions was not reported by the authors. Villanueva et al. [17], for a pH adjusted at 4.5 did not observe any effect on gelatinization temperature for the rice
starch dispersions, whereas the acidification of tapioca starch dispersions facilitated the gelatinization. All these authors agreed that the presence of proteins greatly influenced the gelatinization of these starches in the presence of acetic acid. Indeed, for Ohishi et al., the higher solubility and degradation of rice flour proteins in acetic acid than in distilled water might also accelerate the absorption, swelling, and gelatinization of starch of rice flour [16].

For other acids, the authors found an opposite effect to our results. On wheat starch in presence of HCl ($T_{Ge}$ moved from 58.7 °C to 70.3 °C after 10.5 h [18]), chickpea wet starch with sulfuric acid ($T_{Ge}$ moved from 132.8 °C to 139.6 °C [19]) or quinoa starch treated with $H_2SO_4$ ($T_{Ge}$ moved from 66.7 °C to 86.4 °C [20]). Velasquez-Castillo and al, and Yixiang Xu and al also found an enthalpy increase [19,20]. These changes are attributed to the disappearance of the cooperative melting of the granules, which is facilitated by water uptake in the amorphous parts of the granule [18].

The whole of this work did not lead to the same conclusions, and the effects of acid on starch were sometimes contradictory between several works. The authors' hypotheses expressed that gelatinization enthalpy value, related to the energy required to break down starch organization, could be dependent on crystalline organization, or water intake in amorphous area. It was difficult to compare all the results for two reasons. On the one hand, authors studied starch and not a whole batter and it was shown that ingredient addition could affect starch environment and might interact with acetic acid. Furthermore, the results have been obtained in different pH conditions and pH value could be an important factor to consider. For example, the impact of acetic acid on gelatinization could be related to the electrical potential of flours. Therefore, a study of F20P80 model systems was investigated as a function of pH by µDSC.

For the 3 flours, the $T_{Ge}$ was stable up to a threshold pH (3.4; 4.2, and 4.0, respectively, for wheat, chickpea, and quinoa flour), then dropped when the pH decreased. Figure 2 showed that the starch gelatinization enthalpy in the $F_{w35}P_{65}$ standard mixtures was not influenced by the presence of acetic acid, whereas for the $F_{CP35}P_{65}$ standard mixtures, the evolution of $\Delta H_{Ge}$ followed a “bell-shaped” curve, presenting a maximum enthalpy of 17.6 +/− 1.3 J/g<sub>dry starch</sub> at pH 3.8. Finally, for the $F_{Q35}P_{65}$ mixtures, the enthalpy $\Delta H_{Ge}$ increased significantly from 8.4 +/− 0.3 to 11.7 +/− 0.9 J/g<sub>dry starch</sub> as soon as acetic acid was added, then stabilized.

The isoelectric points (pI) of the different flours were determined by measuring the zeta potential. Indeed, the zeta potential changes from positive to negative values when the pH increases and the pH at which the zeta potential is zero is the isoelectric point. The isoelectric points of the 3 flours ($F_w$, $F_{CP}$, and $F_Q$) were 5.8, 3.7, and 3.0, respectively. The increase in gelatinization enthalpy of $F_{CP}$ flour may be related to the electrical point of chickpea starch. At pHi, the overall charge from surface proteins to starch was zero. If pH < pI, the overall charge was positive. If pH > pI, the overall charge was negative. It was around pH 4 (3.7) that the isoelectric potential of chickpea flour canceled out; this result was also shown by Emami in 2007 [43], leading to potential aggregation. A protein network around the chickpea starch could constitute a steric barrier to the entry of the plasticizer into the grain, which would therefore require more $\Delta H$ energy to gelatinize. These results agree with the hypothesis of Jacobs et al. (1998), which showed lowering the water uptake in the amorphous parts of the granule could increase the gelatinization enthalpy [18]. For wheat flour, the pHi was 5.8, therefore outside the range of pH studied, and for quinoa flour, the pHi was 3, which did not explain the increase in the gelatinization enthalpy at higher pHs. Another hypothesis would be that the action of acetic acid would reveal an attack on the wall of the starch granules.
<figure><img src="image_5.png" /></figure>

<figure><img src="image_6.png" /><figcaption>**Figure 2.** Gelatinization temperatures and enthalpies of standard mixtures as a function of pH.</figcaption></figure>

In order to know the penetration rate of the plasticizer (water + acetic acid) into the starch granule, the curves of cumulative enthalpies were calculated and reported as a function of temperature. The partial gelatinization enthalpy curves for the three starches studied are shown in Figure 3. This study made it possible to observe the kinetics of loss of the ordered structure of starch at a temperature between 45 and 80 °C.
<figure><img src="image_7.png" /><figcaption>**Figure 3.** Effect of acetic acid on cumulated enthalpy for F₃₅P₆₅ mixtures.</figcaption></figure>

At free pH, (5.8; 5.2 and 6.4) the shapes of the curves illustrated for the F₃₅W₆₅ mixtures (Figure 3) were quite similar for the three flours Fw, FCP, FQ. Indeed, the fusion of the ordered zones for FCP and FQ were very close to that of Fw, contrary to that of tubers such as potato, which was much steeper, as reported in the literature (Waigh et al., 2000) [32]. As for the Fw, these observations indicated a de-structuring in two stages (loss of crystallinity and destruction of the helices) and can be partly explained by the surface state of the granules. Fannon et al. (1992) [44] showed that tuber granules appear smooth on the surface under scanning electron microscopy, whereas cereal starch granules such as those of wheat starch had pores of about 100 nm on their surface, generated at the time of granule biosynthesis and offering greater sensitivity to hydration. However, the temperatures at which the granular structure began to be destroyed were higher for chickpea and quinoa. As explained above, this may be due to the amylopectine structure [32], and the significant presence of proteins around the starch granules.

Although at pH 4 (P = water + acetic acid), the penetration rate of the plasticizer remained identical for the F<sub>w35</sub>P<sub>65</sub> and F<sub>CP35</sub>P<sub>65</sub> primary systems (Figure 4), a slight decrease in the slope of the curves were observed for the quinoa flour, which seemed to show that acetic acid attacked the surface of quinoa starch granules more easily. This is in accordance with Velasquez et al. who showed hydrogen bond modification in the amorphous area during quinoa starch acidic treatment [20]. This result explained the increase in the gelatinization enthalpy before a pH of 3 before passing the isoelectric point.

The total gelatinization enthalpy (ΔH) in the presence of acetic acid remained almost constant for the F<sub>w35</sub>P<sub>65</sub> mixture but increased sharply for the F<sub>CP35</sub>P<sub>65</sub> and F<sub>Q35</sub>P<sub>65</sub> mixtures. Our results showed that two different mechanisms were at play to explain the increase in gelatinization enthalpy of the F<sub>CP35</sub>P<sub>65</sub> and F<sub>Q35</sub>P<sub>65</sub> mixtures. Indeed, for chickpea flour, at pH 4, the passage of the isoelectric point led to the aggregation of proteins creating a steric barrier to the plasticizer which caused an increase in enthalpy. For quinoa flour, the considerable increase in the total gelatinization enthalpy suggested a better organization of the ordered regions of the starch, linked to an attack of the amorphous regions by acetic acid. Indeed, according to Donovan's [45] theory, acid hydrolysis preferentially attacks the amorphous regions in the granule, as a result, the crystallites are no longer destabilized by the amorphous parts.
<figure><img src="image_8.png" /><figcaption>**Figure 4.** Superimpositions of the cumulative enthalpy curves for the F₃₅W₆₅ mixtures (a) and the F₃₅P₆₅ mixtures (b).</figcaption></figure>

**4. Conclusions**

In this study, we followed the changes in the gelatinization behavior of three starches (wheat, chickpea, and quinoa) under the influence of acetic acid in vegetable sponge cake in order to obtain new snacks.

The addition of vegetable fibers such as carrots to the sponge cake batter causes a very slight increase in the gelatinization temperature. The quantity of free water available, at this water content, is largely sufficient to allow complete gelatinization without modifying the cooking temperature. On the other hand, the acidification of the sponge cake with white vinegar to reach a pH of 4 causes a modification of its thermal properties. Indeed, the gelatinization of the starch appears at a lower temperature in the sponge cake batter formulated with chickpea and quinoa flour than the starch in the corresponding batter without vinegar. However, it is almost identical to the sponge cake made with wheat flour. This work will allow adjusting the microwave cooking parameters (time and power) to obtain complete gelatinization of these acidified vegetable sponge cakes.
Moreover, it is interesting to observe that the mechanisms of action of acetic acid on starch depend on the type of flour used. Indeed, two mechanisms of action of acetic acid on starch, one direct and the other indirect, have been demonstrated: (1) The acetic acid contained in vinegar acts directly on the starch of quinoa itself at low concentration with a marked increase in the speed of the plasticizer penetration into the granule. (2) For chickpea starch, acetic acid acts indirectly on its gelatinization by modifying the zeta potential of the other flour components, which probably causes steric hindrance for the supply of the plasticizer in the starch granules.

**Author Contributions:** Conceptualization, P.L.-B.; methodology, M.C.; investigation, M.C.; writing—original draft preparation, M.C.; writing—review and editing, P.L.-B.; supervision, P.L.-B., A.R., O.P. and L.V.; reviewing, P.L.-B., A.R., O.P. and L.V.; visualization, M.C.; project administration, P.L.-B.; funding acquisition, O.P. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by NBread® process.

**Institutional Review Board Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** This work was carried out during the PhD project entitled "MACOMO" (Alveolar foam microwave cooking), which was co-funded by N-Bread® process start-up.

**Conflicts of Interest:** The authors do not declare any conflicts of interest.

## References

1. Bonnet, N. *Les Apéritifs à Croquer En Chiffres-2020*; Agence Bergamote: Paris, France, 2020.

2. Businesscoot. *Le Marché Des Biscuits Apéritifs-France*; Businesscoot: Paris, France, 2021.

3. Biolinéaires. Les Biscuits Apéritifs, un Marché de Près de 20 Millions D'euros en Magasin Bio. Available online: https://www.biolineaires.com/les-biscuits-aperitifs-un-marche-de-pres-de-20-millions-deuros-en-magasin-bio/ (accessed on 1 October 2021).

4. Wade, P. Technology of Biscuit Manufacture: Investigation of the Role of Fermentation in the Manufacture of Cream Crackers. *J. Sci. Food Agric.* **1972**, *23*, 1021–1034. [CrossRef]

5. Rogers, D.; Hoseney, R. Effects of Fermentation in Saltine Cracker Production. *Cereal Chem.* **1989**, *66*, 6–10.

6. Doescher, L.D.; Hoseney, R.C. Saltine Crackers: Changes in Craker Sponge Rheology and Modification of a Cracker-Baking Procedure. *Cereal Chem.* **1985**, *62*, 158–162.

7. Desjardins-Lavisse, I.; Olivier, P.; Guillaume, G. Food and/or Nutraceutical Product of the Sponge Cake Type and Method for Obtaining Same from Food. WO 2016/110513 A1, 6 January 2016.

8. Kuda, T.; Yazaki, T.; Takahashi, H.; Kimura, B. Effect of Dried and Vinegar Flavored Squid Products on Acid Resistance of Salmonella Typhimurium and Staphylococcus Aureus. *Food Control* **2013**, *30*, 569–574. [CrossRef]

9. Niaz, K.; Fatima, A. Screening Antibacterial Activity of Vinegar & Olive Oil on Enteric Bacteria. *RADS J. Biol. Res. Appl. Sci.* **2017**, *8*, 14–17.

10. Shah, Q.A.; Bibi, F.; Shah, A.H. Anti-Microbial Effects of Olive Oil and Vinegar against Salmonella and *Escherichia coli*. *Pac. J. Sci. Technol.* **2013**, *14*, 479–486.

11. Guynot, M.E.; Ramos, A.J.; Sanchis, V.; Marín, S. Study of Benzoate, Propionate, and Sorbate Salts as Mould Spoilage Inhibitors on Intermediate Moisture Bakery Products of Low pH (4.5–5.5). *Int. J. Food Microbiol.* **2005**, *101*, 161–168. [CrossRef]

12. Garzon, R.; Skendi, A.; Antonio Lazo-Velez, M.; Papageorgiou, M.; Rosell, C.M. Interaction of Dough Acidity and Microalga Level on Bread Quality and Antioxidant Properties. *Food Chem.* **2021**, *344*, 128710. [CrossRef]

13. Mesías, M.; Delgado-Andrade, C.; Morales, F.J. Alternative Food Matrices for Snack Formulations in Terms of Acrylamide Formation and Mitigation. *J. Sci. Food Agric.* **2019**, *99*, 2048–2051. [CrossRef]

14. Budak, N.H.; Aykin, E.; Seydim, A.C.; Greene, A.K.; Guzel-Seydim, Z.B. Functional Properties of Vinegar. *J. Food Sci.* **2014**, *79*, R757–R764. [CrossRef]

15. Majzoobi, M.; Beparva, P. Effects of Acetic Acid and Lactic Acid on Physicochemical Characteristics of Native and Cross-Linked Wheat Starches. *Food Chem.* **2014**, *147*, 312–317. [CrossRef] [PubMed]

16. Ohishi, K.; Kasai, M.; Shimada, A.; Hatae, K. Effects of Acetic Acid on the Rice Gelatinization and Pasting Properties of Rice Starch during Cooking. *Food Res. Int.* **2007**, *40*, 224–231. [CrossRef]

17. Villanueva, M.; Ronda, F.; Moschakis, T.; Lazaridou, A.; Biliaderis, C.G. Impact of Acidification and Protein Fortification on Thermal Properties of Rice, Potato and Tapioca Starches and Rheological Behaviour of Their Gels. *Food Hydrocoll.* **2018**, *79*, 20–29. [CrossRef]

18. Jacobs, H.; Eerlingen, R.C.; Rouseu, N.; Colonna, P.; Delcour, J.A. Acid hydrolysis of native and annealed wheat, potato and pea starches—DSC melting features and chain length distributions of lintnerised starches. *Carbohydr. Res.* **1998**, *308*, 359–371. [CrossRef]
19. Xu, Y.; Sismour, E.N.; Grizzard, C.; Thomas, M.; Pestov, D.; Huba, H.; Wang, T.; Bhardwaj, H.L. Morphological, Structural, and Thermal Properties of Starch Nanocrystals Affected by Different Botanic Origins. *Cereal Chem.* **2014**, *91*, 383–388. [CrossRef]

20. Velásquez-Castillo, L.E.; Agostinetto Leite, M.; Ditchfield, C.; do Amaral Sobral, P.J.; Freitas Moraes, I.C. Quinoa starch nanocrystals production by acid hydrolysis: Kinetics and properties. *Int. J. Biol. Macromol.* **2020**, *143*, 93–101. [CrossRef]

21. Alibasic, H.; Junuzovic, H.; Selimovic, A.; Selimovic, A.; Brcina, T. Chemical Composition and Sensory Properties of Gluten-Free Crackers with Buckwheat Sourdough. *Int. J. Res. Appl. Sci. Biotechnol.* **2020**, *7*, 108–113. [CrossRef]

22. Murgueytio, E.; Santacruz, S. Volume, Firmness and Crumb Characteristics of Gluten-Free Bread Based on Extruded Quinoa Flour and Lactic Acid. *Braz. J. Food Technol.* **2020**, *23*, e2019220. [CrossRef]

23. Alvarez-Jubete, L.; Auty, M.; Arendt, E.K.; Gallagher, E. Baking Properties and Microstructure of Pseudocereal Flours in Gluten-Free Bread Formulations. *Eur. Food Res. Technol.* **2010**, *230*, 437–445. [CrossRef]

24. Machado Alencar, N.M.; Steel, C.J.; Alvim, I.D.; de Morais, E.C.; Andre Bolini, H.M. Addition of Quinoa and Amaranth Flour in Gluten-Free Breads: Temporal Profile and Instrumental Analysis. *LWT Food Sci. Technol.* **2015**, *62*, 1011–1018. [CrossRef]

25. Miñarro, B.; Albanell, E.; Aguilar, N.; Guamis, B.; Capellas, M. Effect of Legume Flours on Baking Characteristics of Gluten-Free Bread. *J. Cereal Sci.* **2012**, *56*, 476–481. [CrossRef]

26. Sopade, P.A.; Halley, P.J.; Junming, L.L. Gelatinisation of Starch in Mixtures of Sugars. II. Application of Differential Scanning Calorimetry. *Carbohydr. Polym.* **2004**, *58*, 311–321. [CrossRef]

27. Dolores Alvarez, M.; Fuentes, R.; Dolores Olivares, M.; Cuesta, F.J.; Canet, W. Thermorheological Characteristics of Chickpea Flour Slurry as Affected by Moisture Content. *J. Food Eng.* **2014**, *136*, 9–18. [CrossRef]

28. Kaur, M.; Singh, N. Studies on Functional, Thermal and Pasting Properties of Flours from Different Chickpea (*Cicer arietinum* L.) Cultivars. *Food Chem.* **2005**, *91*, 403–411. [CrossRef]

29. Meares, C.A.; Bogracheva, T.Y.; Hill, S.E.; Hedley, C.L. Development and Testing of Methods to Screen Chickpea Flour for Starch Characteristics. *Starch/Staerke* **2004**, *56*, 215–224. [CrossRef]

30. Li, G.; Wang, S.; Zhu, F. Physicochemical Properties of Quinoa Starch. *Carbohydr. Polym.* **2016**, *137*, 328–338. [CrossRef]

31. Ruales, J.; Nair, B.M. Properties of Starch and Dietary Fibre in Raw and Processed Quinoa (Chenopodium Quinoa, Willd) Seeds. *Plant Foods Hum. Nutr.* **1994**, *45*, 223–246. [CrossRef]

32. Waigh, T.A.; Gidley, M.J.; Komanshek, B.U.; Donald, A.M. The Phase Transformations in Starch during Gelatinisation: A Liquid Crystalline Approach. *Carbohydr. Res.* **2000**, *328*, 165–176. [CrossRef]

33. Zhou, Y.G.; Li, D.; Wang, L.J.; Li, Y.; Yang, B.N.; Bhandari, B.; Chen, X.D.; Mao, Z.H. Effect of Water Content on Thermal Behaviors of Common Buckwheat Flour and Starch. *J. Food Eng.* **2009**, *93*, 242–248. [CrossRef]

34. Torres, M.D.; Moreira, R.; Chenlo, F.; Morel, M.H. Effect of Water and Guar Gum Content on Thermal Properties of Chestnut Flour and Its Starch. *Food Hydrocoll.* **2013**, *33*, 192–198. [CrossRef]

35. Chungcharoen, A.; Lund, D.B. Influence of Solutes and Water on Rice Starch Gelatinization. *Cereal Chem.* **1987**, *64*, 240–243.

36. D'Appolonia, B.L. Effect of Bread Ingredients on Starch-Grältinization Properties as Measured by the Amylograph. *Am. Assoc. Cereal Chem.* **1972**, *49*, 532–543.

37. Spies, R.D.; Hoseney, R.C. Effect of Sugars on Starch Gelatinization. *Cereal Chem.* **1982**, *59*, 128–131.

38. Wootton, M.; Bamunuarachchi, A. Application of Differential Scanning Calorimetry to Starch Gelatinization. I. Commercial Native and Modified Starches. *Starch-Stärke* **1979**, *31*, 201–204. [CrossRef]

39. Chiotelli, E.; Pilosio, G.; Le Meste, M. Effect of Sodium Chloride on the Gelatinization of Starch: A Multimeasurement Study. *Biopolymers* **2002**, *63*, 41–58. [CrossRef]

40. Santos, E.; Rosell, C.M.; Collar, C. Gelatinization and Retrogradation Kinetics of High-Fiber Wheat Flour Blends: A Calorimetric Approach. *Cereal Chem.* **2008**, *85*, 455–463. [CrossRef]

41. Goranova, Z.; Marudova, M.; Baeva, M. Influence of functional ingredients on starch gelatinization in sponge cake batter. *Food Chem.* **2019**, *297*, 124997. [CrossRef]

42. Hibi, Y. Effects of Salt, Sugar and Vinegar on Melting of the Amylose-Lipid Complex in Rice Starch. *J. Home Econ. Jpn.* **2002**, *53*, 805–810.

43. Emami, S.S.T. Hydrocyclone Fractionation of Chickpea Flour and Measurement of Physical and Functional Properties of Flour and Starch and Protein Fractions. Ph.D. Thesis, University of Saskatchewan, Saskatoon, SK, Canada, 2007; 347p.

44. Fannon, J.E.; Hauber, R.J.; Bemiller, J.N. Surface Pores of Starch Granules. *Cereal Chem.* **1992**, *69*, 284–288.

45. Donovan, J.W. Phase transitions of the starch-water system. *Biopolymers* **1979**, *18*, 263–275. [CrossRef]