**1. Introduction**

Pumpkin seeds are a source of fat (37–45%) and protein (25–37%), which is distinguished by a high content of indispensable amino acids. They also contain about 11% carbohydrates and 6% dietary fibre, as well as many vitamins and minerals [1]. In Europe, pumpkin (*Cucurbita pepo* L.) is cultivated in the middle south region, primarily for oil production from seed, where the main by-product from oil extraction is defatted pumpkin cake, which contains up to 65% protein [2,3]. Due to the high protein content, this by-product left over from pumpkin oil seed extraction is considered by many authors as one of the most attractive and promising sources of vegetable proteins with proven health-promoting and functional effects [4,5].

One of the uses of pumpkin cake, apart from enrichment with amino acids, is protein preparations constituting newly developed functional ingredients that enrich the nutritional properties of food products. Favourable results have been obtained for extraction of functional proteins from pumpkin seed cakes under alkaline conditions. In this technique, pumpkin cakes are treated with an NaOH solution at pH 8–10, after which the solution is centrifuged and the proteins are precipitated therefrom at the isoelectric point. In this way, isolates containing as much as 80–90% protein can be obtained with high yield. Additionally, this method is used to obtain the broad spectrum of proteins found in pumpkin seeds [5].

Widely available pumpkin protein concentrates and isolates are highly appreciated, despite their poor functional properties. During the manufacture of pumpkin protein

**Citation:** Miedzianka, J.; Zambrowicz, A.; Zieli ´nska-Dawidziak, M.; Drozd˙ z, W.; Nem´s, A. ˙ Effect of Acetylation on Physicochemical and Functional Properties of Commercial Pumpkin Protein Concentrate. *Molecules* **2021**, *26*, 1575. https://doi.org/10.3390/ molecules26061575

Academic Editors: Anubhav Pratap Singh, Przemyslaw Lukasz Kowalczewski and David Kitts

Received: 31 January 2021 Accepted: 10 March 2021 Published: 12 March 2021

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**Copyright:** © 2021 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/).

preparations, the applied heat treatments cause extensive protein denaturation and the resulting preparations are characterized mainly by poor solubility, limiting their incorporation into food systems. Therefore, a roadblock to the large-scale use of plant proteins is their poor solubility under mildly acidic (pH 3–6) conditions. This excludes their use in acidic foods such as coffee whiteners, acidic beverages, yogurts and pourable and non-pourable dressings. The weak solubility of proteins, however, sets limits for their utilization in formulated food systems and so the solubilization of pumpkin protein preparations has been attempted to extend their usefulness in the food industry [6].

One of the most convenient and frequently used methods for altering the functional properties of proteins is acetylation. This chemical modification of proteins involves the reaction of acetic anhydride with the ε-amino group of lysine and other nucleophilic groups such as phenolic (e.g., tyrosine) and aliphatic hydroxyl groups (e.g., serine and threonine) [7]. The acetic anhydride (CAS number 108-24-7) used here is a chemical compound widely used in organic synthesis. It is a colourless liquid that smells strongly of acetic acid. Acetylation of proteins in the range of 0.2–1.0 mL/g protein has been widely known for more than 40 years [8–11]; modified proteins have been applied in the preparation of products such as coffee whiteners [12], flavouring agents for roasted meat [13] and carbonated beverages [14]. Additionally, recently, studies on improving functionality by acylation have been applied to many food proteins including spray-dried egg white [15], oat protein isolates [16] and fish myofibrillar protein [17]. However, in the literature, there is no report available on the functionality of acetylated commercial pumpkin protein concentrate. This chemical modification is considered safe because acetic anhydride is rapidly hydrolysed (half-life 4.4 min) to acetic acid, which is readily biodegradable. In the atmosphere, it is converted to acetic acid, which is subject to photooxidative degradation (half-life 22 days). Toxicity to aquatic organisms is moderate (18 to 3400 mg/L), but it persists only for a short time due to its rapid hydrolysis to acetate/acetic acid. Acetic acid is further used as a food and animal feed additive or a preservative in pickles [18].

For the above-stated reason, this investigation through chemical modification has been undertaken in order to improve the functional properties of pumpkin protein preparations, mainly water-binding, oil-absorption capacities, solubility, foaming and emulsifying properties, while limiting the negative impact on changes in the chemical composition and digestibility of proteins. Therefore, the purpose of the present study was to determine the effect of the anhydride-to-protein ratio of acetylation on the chemical composition and functional properties of commercial pumpkin protein preparations. It is hoped that the data will provide information on acetylated derivatives, which could be useful in experiments concerning the chemical modification of commercial pumpkin protein preparations appropriated for food purposes.
