**Nutraceuticals in Human Health**

Special Issue Editors

**Alessandra Durazzo Massimo Lucarini Antonello Santini**

MDPI • Basel • Beijing • Wuhan • Barcelona • Belgrade • Manchester • Tokyo • Cluj • Tianjin

Italy *Special Issue Editors* Alessandra Durazzo CREA-Research Centre for Food and Nutrition Italy Antonello Santini Department of Pharmacy University of Napoli Federico II Italy *Editorial Office*

MDPI St. Alban-Anlage 66 4052 Basel, Switzerland Massimo Lucarini CREA-Research Centre for Food and Nutrition

This is a reprint of articles from the Special Issue published online in the open access journal *Foods* (ISSN 2304-8158) (available at: https://www.mdpi.com/journal/foods/special issues/ nutraceuticals human health).

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## **Contents**

## **About the Special Issue Editors**

**Alessandra Durazzo** was awarded a Master's degree in Chemistry and Pharmaceutical Technology cum laude in 2003, and a PhD in Horticulture in 2010. Since 2005, she has been a researcher at the CREA-Research Centre for Food and Nutrition. The core of her research is the study of chemical, nutritional and bioactive components of food, with particular regard to the wide spectrum of substances classes and their nutraceutical features. For several years, she was involved in national and international research projects on the evaluation of several factors (agronomic practices, processing, etc.) that affect food quality, the levels of bioactive molecules and the total antioxidant properties, as well as their possible impact on the biological role played by bioactive components in human physiology. Her research activities are addressed also towards developing, managing and updating the Food Composition Database, as well as Bioactive Compounds and Food Supplements databases; particular attention is given towards the harmonization of analytical procedures and classification and codification of food supplements.

**Massimo Lucarini** received a Master's degree in Industrial Chemistry cum laude of the University of Rome "La Sapienza", Italy (1992) and a PhD in Chemistry also of the University of Rome "La Sapienza". His research activity is mainly aimed at the evaluation of nutrient content, molecules with biological and anti-nutrient activity in foods and diets, studies of stability of technological treatments of food products using specific process markers. Particular interest is addressed to the evaluation of the nutritional quality of foods, the bioavailability of nutrients and bioactive components and their interaction with the food matrix (using in vitro models and cellular models), and to applications in the nutraceutical field; recent attention focuses on the exploitation of waste from the agri-food industry, with a view to sustainable agri-food production. In relation to the study of bioactive molecules, the experience gained in this field is wide ranging from carotenoids to phenolic substances, and from caseinophosphopeptides (CPP) to the components of dietary fiber. An integral part of the research carried out is linked to institutional activity, including food composition tables, guidelines for healthy nutrition, and evaluation of fraud risk in the agri-food system. In relation to the production system, the effects of technological treatments on molecules of nutritional interest are also evaluated. He is also interested in using natural substances with strong antioxidant properties to improve the shelf-life of food products. The research activity is also aimed at the development of new analytical methods, the exchange of scientific information and the acquisition of new skills both at the national and international level, through training courses, participation in congresses and seminars. The dissemination activity is carried out through the production of scientific articles, interviews released in national journals and broadcasting systems, the creation of web pages, participation in congresses, educational and informative activities.

**Antonello Santini**, Ph.D., is Professor of Food Chemistry and Food Chemistry and Analysis of Food and Nutraceuticals at the Departments of Pharmacy and at the Department of Agriculture of the University of Napoli Federico II, Napoli, Italy. He is also visiting professor at the Albanian University of Tirana, Albania. He holds a PhD in Chemical Sciences. His research areas of interest are substantiated by many international collaborations, mainly in the field of food; food chemistry, nutraceuticals, functional food; supplements; recovery of natural compounds bioactive using eco sustainable and environment friendly techniques from agro-food byproducts; nanocompounds; nanonutraceuticals; food risk assessment, safety and contaminants; mycotoxins and secondary metabolites; food analysis; chemistry and food education. He is responsible for funded research projects and responsible for general cultural agreements established between the University of Napoli Federico II and many Universities worldwide. His research activity is substantiated by more than 200 papers in peer reviewed reputed international Journals. He is member of the European Food Safety Authority EFSA, ERWG, Parma, Italy; member of the Italian Authority for Food Safety (CNSA), Italian Ministry of Health, Rome Italy; member of Managing Board, Italian Chemistry Society (SCI) Division of Teaching (DD-SCI), Rome, Italy, as well as an expert member for Chemistry, EurSchool, European Commission, Bruxelles, Belgium.

### *Editorial* **Nutraceuticals in Human Health**

#### **Alessandra Durazzo 1,\*, Massimo Lucarini 1,\* and Antonello Santini 2,\***

Received: 13 March 2020; Accepted: 17 March 2020; Published: 23 March 2020

**Abstract:** The combined and concerted action of nutrient and biologically active compounds is flagged as an indicator of a "possible beneficial role" for health. The use and applications of bioactive components cover a wide range of fields, in particular the nutraceuticals. In this context, the Special Issue entitled "Nutraceuticals in Human Health" is focused on the all aspects around the nutraceuticals, ranging from analytical aspects to clinical trials, from efficacy studies to beneficial effects on health status.

**Keywords:** nutraceuticals; bioactive compounds; medicinal food; safety; health; regulation; clinical tests; efficacy; analysis; formulation

#### **Introduction**

The combined and concerted action of nutrient components and biologically active compounds is flagged as indicator of a "possible beneficial role" for health. The use and applications of bioactive components cover a large range of fields, in particular nutraceuticals ones [1–3].

Nutraceuticals are obtained from foods of vegetal or animal origin, and the current interest and ongoing worldwide research aims to shed light and fully clarify their mechanism of action, their safety and efficacy by substantiating their role by means of clinical data [4,5]. An effort to clarify their mechanism of action will in fact open a door to a next generation of therapeutic agents that do not propose themselves as an alternative to drugs, but, instead, can be helpful to: (i) prevent a cluster of conditions that could occur together (metabolic syndrome), e.g. heart disease, stroke, and type 2 diabetes; (ii) to complement a pharmacological therapyespecially for those individuals who do not qualify for a conventional pharmacological therapy [6,7].

This Special Issue is dedicated to the role and perspectives of nutraceuticals in human health, examined from different angles, ranging from analytical aspects to clinical trials, from efficacy studies to beneficial effects on health conditions.

Concerning the study of functional ingredients and applications, Lu Martínez et al. [8] have studied and proposed the use of *Prunus* serotine defatted flour without hydrogen cyanide risk in cookies and protein concentrate in emulsion stability. Ullah et al. [9] well reviewed and summarized the biomedical properties of polysaccharides as therapeutical agents. It is worth mentioning the work of Swat et al. [10] on characterization of fulvic acid beverages available on the global market. Alternative functional ingredients obtained from waste/side products from industrial grape manufacturing, i.e., grape seeds, were investigated by Lucarini et al. [11].

Studies on evaluation of beneficial effects of nutraceuticals in vitro [12] and in vivo [13] models have been presented, in particular dietary supplementation studies in animal [14–16].

At the same time, specific studies on botanicals have been reported by Fredes et al. [17] and Ji et al. [18] focusing on the importance of these vegetal origin sources.

Nutraceuticals are a challenge for the future of prevention and therapy and a triggering tool in medicine area. The possibility to prevent and/or support a pharmacological therapy, which is nowadays mainly based on pharmaceuticals, can be a powerful tool to face pathological, chronic, long-term diseases in subjects who do not qualify for a pharmacological therapy. The big challenge is to improve nutraceuticals bioavailability and clear their mechanism of action adopting nanotechnologies as new delivery approach and clinical studies to assess and detail how they work in detail [19,20]. At the same time, the interest to new food sources and exploring novel nutraceuticals which beyond their nutritional value have also added value as contributing bioactive substances tailored to specific health conditions for a better results in term of efficacy and safety is stimulating interest and research worldwide for new sources and sustainable environmental friendly solutions [21–23].

This Special Issue end point has been to contribute to the growth of this area of research, trigger interest or research on food and add information scientifically substantiated by new data.

We would like to thank all the authors and the reviewers of the papers published in this Special Issue for their great contribution and effort. We are also grateful to the editorial board members and to the staff of the journal for their kind support during the preparation of this Special Issue.

**Author Contributions:** All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication. All authors have read and agreed to the published version of the manuscript.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

© 2020 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 (http://creativecommons.org/licenses/by/4.0/).

## *Article* **Studied of Defatted Flour and Protein Concentrate of** *Prunus serotine* **and Applications**

#### **Analía A. Lu Martínez 1, Juan G. Báez González 1,\*, Minerva Bautista Villarreal 1, Karla G. García Alanis 1, Sergio A. Galindo Rodríguez 2 and Eristeo García Márquez 3,\***

Received: 16 November 2019; Accepted: 23 December 2019; Published: 27 December 2019

**Abstract:** *Prunus serotine* seed, was processed to produce a defatted flour (71.07 ± 2.10% yield) without hydrocyanic acid. The total protein was 50.94 ± 0.64%. According to sensory evaluation of cookies with *P. serotine* flour, the highest score in overall impression (6.31) was at 50% flour substitution. Its nutritional composition stood out for its protein and fiber contents 12.50% and 0.93%, respectively. Protein concentrate (*Ps*PC) was elaborated (81.44 ± 7.74% protein) from defatted flour. Emulsifying properties of *Ps*PC were studied in emulsions at different mass fractions; φ = 0.002, 0.02, 0.1, 0.2, and 0.4 through physicochemical analysis and compared with whey protein concentrate (WPC). Particle size in emulsions increased, as did oil content, and results were reflected in microscope photographs. *Ps*PC at φ 0.02 showed positive results along the study, reflected in the microphotograph and emulsifying stability index (ESI) test (117.50 min). At φ 0.4, the lowest ESI (29.34 min), but the maximum emulsifying activity index (EAI) value (0.029 m2/g) was reached. WPC had an EAI value higher than *Ps*PC at φ ≥ 0.2, but its ESI were always lower in all mass fraction values. *Ps*PC can compete with emulsifiers as WPC and help stabilize emulsions.

**Keywords:** *Prunus serotine*; defatted flour; soluble protein; protein concentrate; emulsifying properties; emulsion stability

#### **1. Introduction**

Nowadays, there is an increasing demand for products of high nutritional quality [1]. Proteins are one of the major components of the human diet because of their nutritional properties. They are also responsible for physicochemical properties such as solubility, water, and oil retention capacity, foaming and emulsifying capacity, viscosity, and gelation, among others. The proteins impact not only the quality of the products, but also acceptance by consumers [2].

Protein is available in a variety of dietary sources [3]. In recent years, the growing concern of consumers with respect to animal safety has forced the industry to use vegetable proteins [4–6]. This type of proteins has health benefits, e.g., reduction of blood cholesterol levels, prevention of obesity and lower risk of heart diseases and cancer [7]. Vegetable proteins, when mixed with cereals, provide an alternative source of amino acids [3], which is why enrichment of other protein sources such as oilseeds and legumes with cereal-based foods has received considerable attention [8].

Baked snacks, such as bread and cookies, are widely accepted and consumed throughout the world and have become an attractive target for feeding and nutritional status improvement programs. This is especially true for cookies, because they not only offer a good vehicle for protein enrichment for consumers, but also because of their wide-spread consumption (5.9 per capita in 2019) and long shelf life [9–11].

The implementation of wheat flour substitutes or mimicry are desirable alternatives to achieve not only a decrease in calories, but also, to obtain a healthier nutritional profile in their composition [12,13]. Legumes and oilseeds such as soy, sunflower, barley, melon seeds, peanuts, hazelnuts, walnuts, sesame seeds, cashews, and almonds, are some alternative sources of flour [9,10].

Also, food grade films, hydrogels, foams, and emulsifiers have been developed from vegetable proteins. Emulsions are capable of absorbing at the oil-in water interface or air-in water dispersion [7,14]. These are part of many processed food formulations. Proteins are widely used for encapsulation of active substances. The proteins are used as a wall material around the active principle droplet, manifesting advantages such as biocompatibility, biodegradability, amphiphilic and hydrophobic and functional properties [15]. Moreover, vegetable proteins can be combined with other polymers, forming a variety of complexes with different structures (e.g., double networks, mosaic textures and cross-linked structures) [7].

In emulsions, the emulsifying activity index, emulsifying stability index, droplet size, interfacial properties and viscosity parameters are used [1]. Other techniques that help to understand the structure of the emulsions and morphology of the particles, particle size, and colloid instabilities (e.g., flocculation, aggregation) are light microscopy, SEM, and dynamic or static light scattering [16]. Among the vegetable proteins emulsifiers options, we found mainly leguminous foodstuffs like soy, lupin, peas, and chickpeas, cereals like wheat, barley, corn, and rice and oil seed such as peanuts, sunflowers, canola, flaxseed, and sesame. [7,17].

In Mexico, the oilseed *Prunus serotine* is widely distributed, and can be found in 16 states of the Republic. Nowadays the production of the fruit goes to 467.96 tons per year [18,19]. However, only the fruit and leaves have been used since colonial times for nourishment and medicinal purposes [20]. While the seed is still of little economic value because of the waste of its nutritional benefits, since it is only consumed as a toasted snack, the main nutrients in its composition are unsaturated fatty acids (89.9%) such as oleic, linoleic, and α-eleostearic acid, crude fiber (10.73 ± 1.49%) and protein (37.95 ± 0.16%) with 88.12 ± 0.72% of digestibility [18,21]. A protein value higher than other oilseeds like *P. dulcis* (19.91%) and *Arachis hypogaea* (22.82%), having lysine as the limiting amino acid. It has also been reported that digestibility values higher than 80% are related to an efficient amino acid bioavailability [18].

Its oil composition is also considered unique because of the significant content of α-eleostearic acid [22]. This acid can be a nutraceutical ingredient because it is capable of providing beneficial health effects, including prevention and/or treatment of a disease [23]. Some studies report that it effectively suppresses growth of cancer cells, lowers serum lipid levels in mammals, and has been proposed as chemotherapeutic agent against breast cancer. *P. serotine* seed oil increase its potential as functional and nutraceutical ingredient [22].

Biotic and abiotic metabolites can contaminate crops and plant-based foods; therefore, toxins must be examined [24]. Cyanogenic glycosides occur in a wide range of food plant species, such as cassava root, apples, lima beans, passion fruit, and almonds [25]. Almonds contain amygdalin as a cyanogenic glycoside (a secondary metabolite) [26]. This metabolite produces hydrogen cyanide (HCN) when it is hydrolyzed. Its effects go from intoxication symptoms to neuropathic problems [27]. Nevertheless, the toasting process to which *P. serotine* seed is subjected as snack, helps to not produce amygdalin because of the temperature it is subjected to. The pericarp of *P. serotine* accumulates amygdalin, but it is acyanogenic because it lacks enzymes to release HCN [28]. In addition, it is devoid of oil content, as well as cyanide components [22]. All these circumstances, along with the fact that the seed has been used for human nutrition since ancient times, allow us to assume that it has little or no toxicity [28].

However, there are some treatments that can reduce or eliminate the risk of poisoning, whereby the focus in on removal of glycoside through washing and/or pressing the food, by enzymatic breakdown of the glycoside, destroying the enzyme or a combination of these methods [29].

From *P. serotine* seed, two valuable products can be obtained, namely α-eleostearic acid with nutraceutical potential applications and the defatted seed with high protein content, which can be used for the development of biscuit products and concentrate protein for the stabilization of emulsions.

We have previously evaluated the study of *P. serotine* oil, so we are focusing on the second product and its derivates. Therefore, the aim was to evaluate *P. serotine* defatted flour without hydrogen cyanide risk in cookies and protein concentrate in emulsion stability.

#### **2. Materials and Methods**

*P. serotine* seeds were obtained from Xochimilco's market in Mexico City, Mexico. Wheat flour (*Triticum* spp.) and canola oil were purchased from a local food store in Monterrey, Nuevo Leon, Mexico. Whey protein concentrate (WPC, MB Pro-mix, 80%) was food grade from Marquez Bros, International, Inc.-whey division, Hanford, CA. Solvents: hexane, *n*-propanol, boric acid, ethanol, phosphoric acid, and hydrochloric acid were of analytical grade (J.T. Baker reagents, Azcapotzalco, Mexico City, Mexico). The reagents sodium chloride, sodium hydroxide, sodium azide, sodium carbonate, sodium tartrate and copper sulphate were purchased from Development of Chemical Specialities in Monterrey, Nuevo Leon, Mexico. Picric acid was from Acce Microbiology in Guadalupe, Nuevo Leon, Mexico, and Coomassie brilliant blue G-250 from ThermoFisher Scientific, Mexico. Sucrose, Tris(hydroxymethyl)aminomethane, SDS, Folin & Ciocalteu's and bovine serum albumin (BSA) were from Sigma-Aldrich, Mexico.

#### *2.1. P. serotine Defatted Flour*

A defatted flour was elaborated from the seeds of *P. serotine* (Scheme 1). Seeds were cracked open with a sterilized metal squeezer, washed with 2.5% NaCl and distilled water (1:5, *w*/*v*) for 30 min with constant magnetic stirring, followed by scalding with hot water at 90 ◦C for 5 min, and drained for 7 min, followed by drying for 1 h at 60 ◦C in an oven with air circulation. Once dried, the oil was removed with a manual oil press (Kinetic, Henan Wecare Industry Co. Ltd., Jiaozuo, China) and the residue (ground seed) toasted to 100 ◦C for 25 min [29]. Subsequently, the remaining oil was removed by constant magnetic stirring with hexane (1: 5, *w*/*v*) to 25 ± 2 ◦C for 1 h. The ground seed was washed and filtered through Whatman paper No. 4 and dried in a hood extractor for 6 h. Finally, it was chopped in a blender and passed through a 70-mesh screen to obtain a *P. serotine* defatted flour [2].

**Scheme 1.** Physical-chemistry process to obtain *P. serotine* defatted flour.

The flour yield was determined by the following formula:

$$\text{Flow weight (\%)} = \frac{\text{Flow weight (g)}}{\text{Seed weight (g)}} \ast 100\tag{1}$$

#### *2.2. Particle Size*

In order to measure the particle size of the *P. serotine* defatted flour, the methodology of Khor et al. [30] was used with some modifications. The flour was measured in a Mastersizer 3000 Hydro LV (Malvern Instruments Ltd., Worcestershire, UK) using the liquid unit. The particle size was evaluated through the volume-weight mean diameter (D4,3) at 25 ± 2 ◦C, as Belorio et al. [31] report. Optical properties of the sample were defined as refractive index 1.37, isopropyl alcohol as dispersant, and an absorption of 0.1. The results were expressed in μm as means ± standard deviation. Wheat flour (*Triticum* spp.) was used as control.

#### *2.3. Chemical Composition*

Analysis were performed on *P. serotine* defatted flour by using Association of Official Analytical Chemistry [32] and compared with wheat flour (control). Moisture, ash, and crude fiber were evaluated gravimetrically (AOAC 14.006, AOAC 925.15 and AOAC 962.09, respectively). The Goldfish method (AOAC 920.36C) was used to determine the fat content. The protein was measured using the Kjeldahl method (AOAC 930.29) and total carbohydrates were determined by the difference using the following equation:

$$\text{HC} \left( \% \right) = \left[ 100 - \left( \text{protein} + \text{lips} + \text{ash} + \text{crude} \,\, \text{liber} \right) \right] \tag{2}$$

#### *2.4. Grignard Test*

To verify that during the process of making *P. serotine* defatted flour, hydrocyanic acid (HCN) was eliminated, a qualitative test was used according to Castro and Rodriguez [33]. Picrosodic papers were prepared and then circles of filter paper (Whatman No. 4) were soaked with 1% picric acid solution and allowed to dry in the dark until they changed color to deep yellow. Once dried, they were impregnated with 10% sodium carbonate and allowed to dry. Afterwards, they were fixed on the lid of amber bottles and two drops of 10% sodium carbonate were added, preventing it from dripping.

The *P. serotine* defatted flour was placed inside the jar to fill a third of it and covered quickly. The bottles were stored in the dark and after 24 h, a reading was taken. As a control, only fractionated *P. serotine* seeds were used (without any treatment). If the paper´s yellow color was maintained or it became light orange, there would be absolutely no problem in its consumption, but if it changed to intense orange or pink, it could only be consumed with caution and if the color became reddish or dark brown, it would not be safe to consume.

#### *2.5. Cookie Preparation*

Four variations of cookie recipe were made according to Jia et al. [34] with some modifications. The cookie dough formula is presented in Table 1. The control recipe was 100% commercial wheat flour (Fc) and the other four were 100, 75, 50, and 25%, respectively, with *P. serotine* defatted flour (F1 to F4, respectively). Butter and sugar were mixed, then creamed with a Kitchen Aid mixer at low speed for one min. Vanilla essence and egg were added and mixed for one minute. In another bowl, all dry ingredients (flour, baking powder, and salt) were sifted and gradually added to the previous mix at low speed for 1 min and then medium speed for one min. When all the ingredients were integrated and homogenized, the dough was wrapped in plastic and allowed to cool at 4 ◦C for 1 h.

**Table 1.** Cookie dough formula.

Fc corresponds to 150 g of wheat flour, F1 corresponds to 150 g of *P. serotine* defatted flour, F2 corresponds to 112.5 g of *P. serotine* defatted flour and 37.5 g of wheat flour, F3 corresponds to 75 g of *P. serotine* defatted flour and 75 g of wheat flour and F4 corresponds to 37.5 g of *P. serotine* defatted flour and 112.5 g of wheat flour.

Once the dough had rested, it was kneaded and spread with a rolling pin and 2 × 2 cm and 0.5 cm high square cookies were cut and, placed in an aluminum tray with waxed paper to prevent them from sticking. The oven was preheated at 160 ◦C for 15 min and the cookies were baked for 12 min at the same temperature. After removal from the oven, the cookies were left to cool at room temperature (25 ± 2 ◦C).

#### *2.6. Sensory Evaluation and Chemical Composition*

The evaluation was carried out in the Sensory Evaluation Laboratory of the Faculty of the College of Food Science at the Autonomous University of Nuevo Leon, Mexico. Fifty-five panelists (untrained) participated in the sensory test based on Jia et al. [34]. These individuals were seated at individual tables in different compartments. A 9-point hedonic scale was used (1 = extreme dislike, 5 = neither like nor dislike, 9 = extreme like) to evaluate the cookies texture, appearance, color, smell, taste, mouthfeel, aftertaste, and overall impression. Scores of five and higher for overall impressions were considered acceptable in this study. Cookies with 3-digit random number codes were randomly presented to the panelists, who were instructed to cleanse their palates with distilled water (25 ◦C) between sensory analyses. Chemical composition analysis (fat, protein, crude fiber and carbohydrate) involved quantification in the cookies with the highest score for the overall impression attribute, as specified in Section 2.3.

#### *2.7. Extraction of Soluble Proteins*

Proteins were extracted sequentially from *P. serotine* defatted flour according to the procedure described by Ramirez Pimentel et al. [35] and Raya Perez et al. [28], with the following solvents: distilled water (albumins), 0.5 M NaCl solution in 50 mM Tris pH 8 (globulins), 55% (*v*/*v*) 2-propanol (prolamins) and 0.1 M boric acid with 0.5% SDS pH 8 (glutelins).

The flour:solvent mixture (ratio 1:10, *w*/*v*) was stirred for 1 h at 25 ± 2 ◦C. The extracts were centrifuged (Hermle Z326, Labortechnik GmbH, Wehingen, Germany) at 13,000 *g* at 25 ± 2 ◦C for 20 min and the supernatants filtered (Whatman No. 4). The extraction with each solvent was repeated on the same sample sequentially and the supernatants of the three extractions were combined.

#### *2.8. Soluble Protein Determination*

Soluble proteins were quantified from the soluble protein extractions as reported by Lopez Dellamary Toral [36] based on the Bradford [37] technique, with some modifications. Bovine serum albumin (BSA) was used as a standard (0.05 to 0.5 mg/mL). The soluble protein fractions were diluted with 50 mM Tris-HCl buffer at pH 7, to obtain values within the standard range concentration. Albumin concentration was 0.49 mg/mL, globulin 0.26 mg/mL, prolamin 0.33 mg/mL, and glutelin 0.24 mg/mL. In microplates, 20 μL of each extract was added in triplicate, using wells consecutively with 200 μL of Bradford reagent (0.01% Coomassie Blue G-250, 4.75% ethanol, 85% H3PO4), allowing to stand for 2 min. The samples were evaluated (microplate reader-Anthos 2020 version 2.0.5) at 620 nm.

#### *2.9. Electrophoresis*

Protein patterns were analyzed according to Syros et al. [38] with some modifications based on Bio-Rad laboratories [39] using polyacrylamide gel electrophoresis (SDS-PAGE). Two glass plates were placed in the electrophoresis chamber, fixing them with plastic spacers and polyacrylamide gel (4–20%) with a 10-well comb (Mini-PROTEAN TGX, Precast protein gels, Bio-Rad Laboratories, Inc. Irvine, CA, USA).

In gel rails, 20 μL of each extraction of soluble protein fraction were placed (at the same previous concentrations) with distilled water. After electrophoresis, the gel was completely immersed in a fixing solution and washed three times for 10 min with distilled water. The gel was immersed and stirred in Coomassie blue dye solution (G-250) until bands were clearly evidenced.

#### *2.10. Isoelectric Point (pI)*

The isoelectric point of the *P. serotine* defatted flour was determined according to the theoretical determination of proteins and other macromolecules, through zeta potential (ζ-potential) which is the most direct characterization of the repulsion or attraction strength between their acid-base residues [40,41]. For this, a mixture of flour: deionized water in a 1:20 ratio (*w*/*v*) at different pH with 0.1 N NaOH and 0.1 N HCl was vortexed for two minutes. Zetasizer Nano ZS90 light scattering equipment (Malvern Instruments, Worcestershire, England, UK) was used. The measures were in automatic mode using a universal immersion cell (ZEN 1002, Malvern Instrument, Worcestershire, UK) at 25 ◦C. The results were reported as the average of three separate injections, with three measures per injection. The averages of triplicate values were used as the values for zeta potential reported.

#### *2.11. Prunus serotine Protein Concentration (PsPC)*

To obtain *Ps*PC the results obtained from the p*I* were taken as a basis. Variations of the procedure were undertaken to determine the one that was repeatable and had protein concentrate values ≥ 80% and ≤ 90%. All procedures were initiated by mixing the *P. serotine* defatted flour for 1 h in vortex with distilled water at pH 11 with 0.1 N NaOH (25 ± 2 ◦C), ratio 1:20 (*w*/*v*). Then the sample was isolated by centrifugation (Hermle Z326, Labortechnik GmbH, Wehingen, Germany) at 13,000 *g* for 30 min and filtered through No. 4 Whatman paper to obtain two fractions (residue and supernatant).

In the first variation, up to two extractions of the residue obtained in the first part of the process were carried out with 5% NaCl (1:20, *w*/*v*), at two extraction times (30 min and 1 h) in vortex at 25 ◦C, followed by centrifugation (Hermle Z326, Labortechnik GmbH, Wehingen, Germany) at 13,000 *g* for 30 min and filtered, again obtaining two fractions. The residue was analyzed utilizing the Kjeldhal method [32] to ensure the lowest protein loss in the process. The resulting supernatant was combined with the supernatant obtained in the first part, to subsequently acidify and solubilize the protein with HCl as shown in Scheme 1. The precipitate was stored until analysis at −20 ◦C. The pH for acidification were 3.0, 3.7, and 4.5.

In the second variation, the residue of the first part was automatically discarded and the supernatant was acidified with HCl and left to rest for 30 min. Finally, it was centrifuged and filtered under the previous conditions. The precipitate was collected and stored at −20 ◦C until use. Three acid pH values (3.0, 3.7, and 4.5) were tested (Scheme 2).

All final precipitates were analyzed according to proximal analysis via the Kjeldhal method based on AOAC 930.29 [32]. The yield was determined by the following equation:

$$\text{Protein concentration yield (\%)} = \frac{\text{Principalate weight (g)}}{\text{Defeated flow weight (g)}} \text{ to 100} \tag{3}$$

**Scheme 2.** The diagram in gray indicates the procedure for obtaining protein concentrate from the *P. serotine* defatted flour with NaCl treatment and acid pH. 1× indicating that the extraction was performed once, and 2×, that it was repeated twice. The black dots indicate the procedure for obtaining protein concentrate from the *P. serotine* defatted flour by direct acidification.

#### *2.12. Preparation of Emulsions*

The emulsifying agents (*Ps*PC and whey protein concentrate) were prepared at 1% *w*/*v* in deionized water and solubilized with constant stirring. Then the sample was allowed to hydrate overnight at 4 ◦C. Afterwards, 0.05% sodium azide was mixed to prevent microbial growth. Different amounts of canola oil 0.1, 1, 5, 10, and 20 g were added, to obtain a variety of mass fractions (φ = 0.002, 0.02, 0.1, 0.2, and 0.4). The emulsions were mixed in a homogenizer (OMNI International GLH, Georgia, United States) at an initial speed of 1000 rpm for 2 min and subsequently at 3000 rpm for 3 min. All emulsions were made in triplicate and stored at 25 ± 2 ◦C for 18 days, and every three days, all the following analyzes were made. Whey protein concentrate (WPC) was used as a control [42,43].

#### *2.13. Droplet Size Measurement*

Particle size was determined by integrated light scattering using a Mastersizer 3000 Hydro LV (Malvern Instruments Ltd., Worcestershire, UK). The emulsions were analyzed immediately after preparation in quintuplicate. Laser diffraction measures the particle size distribution (diameter equivalent to the volume) from the angular variation of the intensity of scattered light when the laser beam passes through the particles dispersed in solution. The data are then integrated based on the angular dispersion intensity, calculating the particle size through the Mie theory of light scattering. The droplet size of emulsions was evaluated through volume-surface mean diameter (D3,2) at 25 ± 2 ◦C as Guo and Mu reports [1]. Optical properties of the sample were defined as refractive index 1.43 for *Ps*PC and 1.46 for WPC, water as dispersant and an absorption of 0.1. The results were expressed as means ± standard deviation [30].

#### *2.14. Emulsifying Activity Index and Emulsifying Stability Index (EAI and ESI)*

The EAI and ESI were assayed via the colorimetric method, previously reported by Guo and Mu [1]. Immediately after homogenizing each emulsion, 20 μL from the bottom was taken and diluted with 5 mL of 0.1% SDS solution. It was vortexed for 5 min and, the absorbance was measured in a spectrophotometer (UV-Visible-Genesys 10s, Thermo scientific, Cambridge, MA, USA) at 500 nm [1]. The *EAI* and *ESI* were calculated using the following equations:

$$EAI\left(\frac{m^2}{g}\right) = \frac{2 \text{ \* 2.303 \* } A\_0 \text{ \* dilution factor}}{\text{c \* 1 \* } (1 - \phi) \text{ \* 10,000}}\tag{4}$$

where *c* is the initial protein concentration which is 1% *w*/*v*, φ is the oil weight fraction, dilution factor was 250.

$$ESI \left(\text{min}\right) = \frac{A\_0}{A\_0 - A\_{10}} \ast \text{ } t \tag{5}$$

where *A*0 and *A*10 are the absorbance of the diluted emulsions at 0 and 10 min, respectively and, *t* was 10 min.

#### *2.15. Interfacial Protein Concentration*

According to Eichberg and Mokrasch [44] and Guo and Mu [1], interfacial protein concentration was quantified. Two milliliters of freshly prepared emulsions were diluted with 2 mL of 50% sucrose solution (*w*/*v*) and vortexed for 5 min at 25 ± 2 ◦C. In a centrifuge tube, 2 mL of the solution were mixed with 7 mL of 5% sucrose solution (*w*/*v*) and all samples were centrifuged (Spectrafuge 6C, Labnet International, Inc., New York, NY, USA) at 3500*g* for 30 min at 25 ± 2 ◦C. Once centrifuged, three phases were observed: the oil drops in the upper phase, an intermediate phase corresponding to the 5% sucrose solution, and the aqueous phase in the lower part of the tube. The tubes were frozen at −40 ◦C for 24 h and then the upper layer of the oil was removed.

The proteins adsorbed from the oil phase were removed by adding 20 mL of 1% SDS (*w*/*v*) solution. To determine the concentration of adsorbed protein, 1 mL of the sample was mixed with 3 mL of an alkaline copper reagent (A: 2% Na2CO3, 0.4% NaOH, 0.16% sodium tartrate, and 1% SDS with B: 4% CuSO4. 5H2O, in a ratio of 100:1). The samples were vigorously stirred, and allowed to rest at 25 ± 2 ◦C for 10 min. Subsequently, 0.3 mL of 2 N Folin-Ciocaletu was added and allowed to stand for 45 min at 25 ± 2 ◦C. The absorbance was immediately measured at 660 nm in a spectrophotometer (UV-Visible-Genesys 10s, Thermo scientific, Cambridge, MA, USA) against a blank. Bovine serum albumin (BSA) was used as a standard. The interfacial protein concentration was calculated as:

$$T\left(\frac{mg}{m^2}\right) = \mathbb{C}\_{\text{ad}} / \mathbb{S}\_V \tag{6}$$

where *Cad* (mg/mL) is the concentration of adsorbed protein and *SV* is the specific interfacial area (m2/mL emulsion) of the emulsion droplets.

#### *2.16. Optical Microscopy*

The optical microscopy photographs were taken based on Huang et al. [45] with small modifications. Emulsions were mixed in vortex for 1 min prior to analysis. A drop of the emulsion was placed between

the coverslip and microscope slide. The globules of the emulsions were examined and observed under bright field illumination with 40× objective lens on a Leica microscope (Leica DM500, 9435 Heerbrugg, Switzerland) along with the software Leica LAS EZ 2.0.0, Ltd., Application Suite (Leica Microsystems, 9435 Heerbrugg, Switzerland).

#### *2.17. Statistical Analysis*

Data from the replicated experiments were analyzed to determine whether the variances were statistically homogeneous, and the results were expressed as the mean ± standard deviation (SD). Statistical comparisons were made by one-way variance analysis (ANOVA) followed by Tukey's test using Statgraphics centurion XVII Software. The difference between means was considered significant at *p* < 0.05.

#### **3. Results and Discussion**

#### *3.1. Particle Size of Defatted Flour*

*P. serotine* defatted flour had a yield of 71.07 ± 2.10%. Its particle size (D4,3) was 5.10 ± 0.03 μm, which was minor for the commercial wheat flour (*Triticum* spp.) with 7.31 ± 0.01 μm, making *P. serotine* flour 1.4 times smaller.

The AOAC 965.22 [46] mentioned that wheat flour must be able to pass through a No. 70 mesh (212 μm) to be acceptable commercially, and *P. serotine* flour in the process of elaboration did pass through this mesh screen and its particle size was smaller than that of wheat flour (control).

It is already known that the particle size of wheat flour can influence cookie quality, but this can also be true for gluten-free flours. Belorio et al. [31] observed that cookies with smaller values of the elastic component (*G*') correspond to flours with higher values of D4,3. Meanwhile, the biggest elastic values (*G*') were found in doughs elaborated from finer flours.

#### *3.2. Chemical Composition*

The moisture in both flours passed the test mentioned in Codex Standard 152-1985 [47]. *P. serotine* defatted flour was 4.6 times less moisture than wheat flour (Table 2). It was not possible to remove all the oil contained in the *P. serotine* seed. After the separation process, 3.4% was quantified. Possibly because of the oil being bound to proteins contained in *P. serotine*, it also contains 5 times more protein and 1.5 times as much total fiber than wheat flour and 2 times less carbohydrates [48].

The values are the average of five assays ± standard deviations of the flours. Mean values labeled with a different letter in the same file are significantly different (*p* < 0.005). 1 Wheat nutritional values were consulted on https://fdc.nal.usda.gov/fdc-app.html#/food-details/168936/nutrients.

#### *3.3. Grignard Test*

Zumaeta Cordova and Gonzales Díaz [29] mentioned that the treatments that allow the release of hydrocyanic acid from glycosides and their subsequent elimination by drying or heating, are those that guarantee greater safety (100 ◦C for 25 min).

The experiment showed that in the control sample (fractionated seed without treatment), the paper impregnated with picric acid changed from yellow to deep orange, which indicated that the food could be consumed, but with caution. Meanwhile, the flour with treatment paper remained with the same yellow color. This indicated that there were no potential consumption problems (Figure 1).

**Figure 1.** (**a**) *P. serotine* seed without treatment. (**b**) *P. serotine* defatted flour with treatment.

We believe that due to the low concentration of cyanogenic compounds, the consumption of *P. serotine* seeds has not caused any poisoning problems. In addition, the seed has been toasted for a long period of time prior to consumption.

According to Alveano Aguerrebere [20], there is no significant difference between the protein content of the seed in its toasted version (37.95 ± 0.16%), compared to when it is raw (36.55 ± 0.22%). Also, Garcia Aguilar et al. [18] mentioned that there is no significant difference in the values of in vitro protein digestibility of raw (88.12 ± 0.72%) and toasted (89.40 ± 1.32%) *P. serotine* seeds.

#### *3.4. Sensory Evaluation*

The effects of the addition of *P. serotine* defatted flour are shown in Table 3. A decrease in the scores of all sensory attributes and the overall impression of the cookies was found with the addition of *P. serotine* defatted flour. However, the maximum amount of *P. serotine* defatted flour accepted by the untrained panelists in the cookies was 75% (F2) based on the value obtained in the overall acceptability category though the formulation with *P. serotine* defatted flour showing the highest acceptability level was F3 with a substitution of 50%.

**Table 3.** Effects of *P. serotine* defatted flour on sensory attributes of cookies.

The values are the average of fifty-five assay ± standard deviations of the sensory attributes of cookies with *P. serotine* defatted flour and wheat flour. Mean values labeled with a different letter in the same file are significantly different (*p* < 0.005).

The results obtained in the evaluation show that the cookies that were made with 75 to 25% *P. serotine* defatted flour (F2 to F4) received a score greater than five in overall impression, making them acceptable. These cookies had greater acceptance compared to other cookies made with Californian almonds (*P. dulcis*), for which maximum acceptance was only 20% substitution, with a score of 5.25 in overall impression [34].

#### *3.5. Cookie Chemical Composition*

Based on the results obtained from the sensory test, a decision was made to carry out the chemical analysis on Fc cookies (100% wheat flour) because it was preferred by the evaluators. Of the cookies made with *P. serotine* defatted flour, those substituted by 50 and 25% (F3 and F4) were selected as a result of obtaining the highest score in overall impression (Table 4).

**Table 4.** Nutritional components in cookies with wheat and *P. serotine* defatted flour.

The values are the average of three assays ± standard deviations of cookies with wheat and *P. serotine* defatted flour. Mean values labeled with a different letter in the same file are significantly different (*p* < 0.005).

Cookies made with *P. serotine* defatted flour stood out for having fiber and for their protein content, up to 17 times higher than cookies with wheat flour (Fc), as well as, for presenting 6.57 and 2.35% lower carbohydrates, and 6.14 and 9.6% lower fat content (F3 and F4, respectively) than control cookies. In addition, it can be said that cookies made with 50 and 25% *P. serotine* defatted flour have a lower gluten content compared to control cookies, since almonds are the best vegetable sources of gluten-free protein and one of the most popular ingredients in the preparation of gluten-free foods, making them a healthy alternative for people suffering from celiac disease [49].

#### *3.6. Soluble Protein Determination*

From the *P. serotine* defatted flour 16.4 ± 2.54 g soluble protein was extracted/100 mL of solution, which is equivalent to 32.15 ± 0.49% of total protein content. The soluble protein profile was albumin 76.95 ± 2.29%, globulin 13.60 ± 2.56%, glutelin 6.16 ± 0.99%, and prolamin 3.29 ± 0.37%. The relative concentration of soluble protein with respect to insoluble proteins was 3.3:1 (Table 5). Raya Perez et al. [28], also quantified soluble protein in *P. serotine* and also reported albumin as the predominating fraction, followed by globulin, glutelin, and finally prolamin.

**Table 5.** Soluble protein content in *P. serotine* defatted flour.

The values are the average of three assays ± standard deviations of the soluble proteins in *P. serotine* defatted flour. Mean values labeled with a different letter in the same column are significantly different (*p* < 0.005).

#### *3.7. Electrophoresis*

The SDS-PAGE patterns for *P. serotine* defatted flour is reported in Figure 2. The molecular weight of albumin varied in a range from 63 to 20 KDa (lane 2 and 6). In globulins, it varied between 63 and 20 KDa (lane 3 and 7), in prolamins, it ranged from 60 to 20 KDa (lane 4 and 8), and in glutelins from 60 to 12 KDa (lane 5 and 9).

**Figure 2.** Soluble protein patterns of *P. serotine* defatted flour. 1. Molecular weight markers, lanes 2 to 5 (without 2-mercaptoethanol); 2. albumin; 3. globulin; 4. prolamin; 5. glutelin. Lanes 6 to 9, as lanes 2 to 5 but with 2-mercaptoethanol.

The molecular weights obtained were similar to the ones reported by Raya Perez et al. [28]. Albumin weight varied between 65 and 20 KDa, globulin between 65 and 14 KDa and prolamins and glutelins between 65 and 12 KDa, respectively.

Albumins and globulins are the main storage proteins of dicotyledonous plants (e.g., legumes and oilseeds), whereas prolamins and glutelins predominate in monocotyledonous plants (e.g., cereals). As expected of a nitrogen source, storage proteins are rich in asparagine (and aspartate), glutamine (and glutamate), and arginine [50], which is the case of *P. serotine* seed. According to Garcia Aguilar et al. [18] the seed contains 116.97 mg/g of asparagine, 273.73 mg/g of glutamine, and 87.42 mg/g of arginine (toasted version), the three amino acids showing the highest values.

Sze Tao and Sathe [2] have reported that pepsin is the most efficient protease hydrolyzing almond (*P. dulcis*) protein, especially for polypeptides with molecular weights from 15 to 42 KDa. Typically, pepsin hydrolysis produced polypeptides with 15 to 36 KDa, followed by 15 to 20 KDa and some with 20 to 40 KDa. Therefore, *P. serotine* defatted flour protein may be useful in production of food protein hydrolysate and did not necessarily involve an additional process.

#### *3.8. Isoelectric Point (pI)*

The isoelectric point of an amino acid is the pH value at which the amino acid is doubly ionized or in zwitterion concentration and is deduced from the Henderson–Hasselbach equation, as the average of the p*K* values of the stages that form and decompose the zwitterion. The point of intersection of calibration curve with the x-axis is p*I* value of protein [34,51].

As a result of the conductivity measurement at different pH of the *P. serotine* defatted flour, it was found that the specific p*I* for this oilseed was 3.7 (Figure 3). This value can be attributed to the high content of acidic amino acids present in the oilseed (aspartic acid 112.29 mg/ g and glutamine 256.84 mg/g), which influenced the low value of p*I* [17]. In addition, this value is within the optimum range for the precipitation of oil proteins such as peanuts (4.0 ± 0.25), coinciding with what other researchers have reported [52].

**Figure 3.** Isoelectric point (p*I*) of *P. serotine* defatted flour as a function change of concentration of hydrogen concentration.

#### *3.9. Prunus serotine Protein Concentration (PsPC)*

The *P. serotine* defatted flour was subjected to different treatments to obtain a process that is repeatable, standardized, and therefore reliable. The processes were adjusted and carried out as the results were obtained.

Usually to solubilize protein from oilseed meal, alkaline solutions are used. Solutions with a pH of 9 to 12 have higher protein yields. However, in values of pH 12 and higher, isolates with better quality are not always obtained [53].

The salts increase the solubility through the salting-in process, whereby the counter ions cover the ionic charges of protein molecules [54]. NaCl is a solubilizing agent, and the combination of alkali and salt is often used to improve protein solubility [53]. Table 6 shows the results of treating the flour with an alkaline pH followed by the interaction with a saline solution at different numbers of extractions in order to extract and recover the highest protein content of the first residue of flour.

The values are the average of three assays ± standard deviations of protein extractions from *P. serotine* defatted flour. Mean values labeled with a different letter in the same file are significantly different (*p* < 0.005).

The sample subjected to two extractions of one hour each with NaCl at a 1:20 (*w*/*v*) ratio at pH 11 had the lowest remaining protein in the residue (13.30 ± 0.39%), which would indicate that this process allows the collection of more protein in the supernatant of the treated sample.

The most common approach to recover solubilized proteins is by precipitating it with pH adjustment. In peanuts, some authors use pH 4.5 [53,54], while other researchers mention that the optimum pH can be within the isoelectric region between pH 3.0 and 5.0 [47]. Based on all these data, the following pH were used to precipitate the proteins of the supernatants: 4.5, 3.7, and 3.0.

The final protein content precipitated from the collected supernatant was not enough to reach the desired value of protein concentrate (whey protein concentrate ≥ 80%). The resulting values were: 56.77 ± 9.20 (pH 4.5), 66.96 ± 1.21 (pH 3.7), and 72.02 ± 5.01 (pH 3.0). This was attributed to the fact that at a high ionic strength, proteins can be almost completely precipitated from their solution because of dehydration in the protein molecules, thus reducing their solubility [53]. Therefore, the number and time of extraction was reduced in this, as well as in the flour:NaCl ratio (Table 6). At the same time, tests were carried out on the *P. serotine* defatted flour involving interaction only with an alkaline solution at pH 11 and then the supernatant was acidified to identify, in which acidic pH was the most effective.

The results obtained when precipitating the supernatants at pH 3.0 compared to other pH values (3.7 and 4.5) yielded a higher protein percentage. This was corroborated with the direct acidification process, which showed a concentrate value of 81.99 ± 6.96%.

The procedure was repeated two more times and results show that the treatment with direct acidification was the most effective. The average value of protein concentration in the final *P. serotine* defatted flour precipitate was 82.0%.

Researchers have identified and quantified the amino acids present in *P. serotine* seed, as well as its total and soluble protein [18,20,40]. However, there are still no reports on the uses or applications of *P. serotine* protein concentrate, making this work one of the first in its findings.

#### *3.10. Droplet Size Measurement of Emulsions*

In Figure 4, it can be seen that emulsions with more oil content had the largest droplet size. As the days passed, the particle size increased when φ ≥ 0.2. In every *Ps*PC emulsion, the maximum droplet size value was reached at different days, but it could be considered as average on day 10. While in WPC emulsions, the range of days to reach the maximum droplet size was more stable (between days 6 and 12), the particle size was more dispersed compared to the *P. serotine* protein.

**Figure 4.** Droplet size (D3,2) of emulsions with (**a**) protein concentrate (*Ps*PC) (1% *w*/*v*) compared to (**b**) whey protein concentrate (WPC) (1% *w*/*v*) at different φ.

The *Ps*PC emulsion at φ 0.02 presented a droplet size of 4.39 ± 0.08 μm as a maximum, becoming the smallest and more constant emulsion during the time of experiment, and for WPC, it was at φ 0.002, with a value of 2.20 ± 0.20 μm. In both the control (WPC) and *Ps*PC emulsions, on the other hand, at φ 0.4, the droplet size had the highest value of 6.88 ± 0.11 μm (day 9) and 14.36 ± 0.31 μm (day 15), respectively, during storage time because of coalescence.

A small droplet size is of interest in emulsion studies, because they are strongly correlated with high emulsion stability [16]. Authors such as Pandolfe [55] and Floury et al. [56] have reported that the increase of oil in emulsions led to a gradual increase of oil droplet sizes. Part of the effect may be due to the limitation of surfactants in emulsions, since as the oil content increases, the available proteins decreases, limiting the stabilizing benefits of the protein, thus favoring the coalescence of the oil drops, and therefore, increasing the diameter. We suggest that emulsion stability is due to the hydrophobicity of the polypeptide chain. The mean diameter of the droplets in food emulsions can vary from less than 0.2 μm (for cream liqueurs) to greater than 100 μm (for salad dressings), depending on the product [57].

#### *3.11. Emulsifying Activity Index and Emulsifying Stability Index (EAI and ESI)*

Compared with other particles, the protein particles have emulsifying properties and great potential to form soft particles [42]. The ability of a protein to form an emulsion can be defined as an emulsifying activity index (EAI), which determines the approximate amount of interfacial area that can be stabilized per unit amount of protein. Additionally, the stability of the emulsion over a specific time period is referred to as the emulsifying stability index (ESI) [58]. The EAI increased as did the mass fraction in *Ps*PC emulsions (Figure 5). The effect was similar in the control emulsions.

**Figure 5.** (**a**) Emulsifying activity index (EAI) and (**b**) emulsifying stability index (ESI) of *Ps*PC emulsions compared with WPC at different φ.

On the contrary, the stability time diminished as the mass fraction (φ) increased in ESI (Figure 5). In *Ps*PC emulsions, ESI went from 117.50 ± 2.17 (φ = 0.002) to 29.34 ± 1.48 min (φ = 0.4) and in WPC emulsions, from 95.83 ± 7.95 (φ = 0.002) to 19.87 ± 1.08 min (φ = 0.4). For the control emulsions, less stability time was always reported compared to those of *Ps*PC.

Different authors have mentioned similar characteristics in almond proteins, wheat gluten, and acidic subunits of soy (11S globulin) [2,59]. Guo and Mu [1] also got similar results when they studied emulsifying properties of sweet potato protein and found that at low protein concentrations (<1%, *w*/*v*), the EAI values are greater, because it facilitates the formation of new droplets, and with the increase of oil, ESI value decreases. Nevertheless, at oil volumes >35% *v*/*v*, there is a marked increase in ESI, a phenomenon that has been reported also by Sun and Gunasekaran [60] for whey protein isolates.

EAI can be related with interfacial effect and low interaction with aqueous solutions. Our previous droplet size results can be associated with the EAI; as these indexes increased, the droplet size also increased. This can be attributed to the proteins that are surface active molecules with the capacity to improve the stability of oil-in-water emulsions, creating a protective membrane that generate repulsive interactions between oil droplets [16].

#### *3.12. Interfacial Protein Concentration*

The effect of interfacial protein concentration shows the oil drops phase separation after being centrifuged. The values suggest that emulsions were stable. When values of φ < 0.2 were used, it was difficult to separate the phases and reported the values.

Table 7 shows the results of emulsions with φ 0.2 and 0.4. Control emulsions with WPC showed that as the volume of oil increases, the protein content at the interface is diminished.

**Table 7.** Interfacial protein concentration in *Ps*PC and whey protein concentrate (WPC) emulsions.

The values are the average of three assay ± standard deviations of interfacial protein concentration in *Ps*PC and WPC emulsions.

The interfacial protein concentration in control emulsions was more constant, in contrast with *Ps*PC, which showed a higher value as oil volume increased, allowing more stability.

#### *3.13. Optical Microscopy*

The microphotographs in function of *Ps*PC and WPC are shown in Figures 6 and 7, respectively. The images reflect the results of droplet size analysis. It shows that as the mass fraction of emulsions increased, the droplet size also decreased and began to show coalescence. Guo and Mu [1] report similar results. The maximum droplet size can be seen between day 9 and 12 in both emulsions.

**Figure 6.** *Cont.*

**Figure 6.** (**a**–**e**) Microphotographs of *Ps*PC emulsions at 40×. Microphotographs show particle size in function of oil in aqueous phase.

**Figure 7.** *Cont.*

(**e**) WPC φ 0.4

**Figure 7.** (**a**–**e**) Microphotographs of WPC emulsions at 40×. The particle size changes depending on the oil phase increase.

WPC and *Ps*PC emulsions at their smallest mass fractions (φ = 0.002 and φ = 0.02) showed more homogenization.

#### **4. Conclusions**

The *P. serotine* seed is an important source of protein and mainly contains albumin. *P. serotine* defatted flour can be used to replace wheat flour at 50% in the preparation of cookies with acceptable sensory property. We believe that it is necessary to remove all cyanogenic compounds in flour of *P. serotine*. The Grignard test shows a positive reaction in flour without thermic treatment. The content of raw fiber in cookies was almost the same in all treatments. *P. serotine* flour purification can be a concentrated protein source with possible applications to stabilize emulsions.

The alkaline (pH 11) and acid (pH 3.0) process showed a higher concentration of protein than the ionic force process as a function of the sodium chloride concentration. The protein concentrate is comparable to WPC in that it forms stable emulsions with oil content of less than 20% by weight, without changing the particle size during 18 days of storage.

**Author Contributions:** A.A.L.M., J.G.B.G., E.G.M., K.G.G.A. conceived and designed the experiments; A.A.L.M. and M.B.V. performed the experiments; J.G.B.G., A.A.L.M., M.B.V., K.G.G.A., S.A.G.R., and E.G.M. analyzed the data; J.G.B.G., K.G.G.A., and E.G.M. helped with reagents/plant materials/analysis tools; and A.A.L.M., M.B.V., J.G.B.G. and E.G.M. were involved in drafting the manuscript and revising it. All authors have approved the final version. All authors have read and agreed to the published version of the manuscript.

**Funding:** We thank CONACyT for the financial support through the project PN-2015-01-1470 and UANL-PAICyT CT723-19.

**Acknowledgments:** We would like to thank Consejo Nacional de Ciencia y Tecnología (CONACyT) for financially supporting through the project Problemas Nacionales-2015-01-1470 and financially supporting A.A.L.M. to obtain her Ph.D. (scholarship 611290), and Universidad Autónoma de Nuevo León (UANL) for financially supporting through the project PAICyT CT723-19.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analysis, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

#### **References**

© 2019 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 (http://creativecommons.org/licenses/by/4.0/).

## *Review* **Sources, Extraction and Biomedical Properties of Polysaccharides**

#### **Samee Ullah 1,2, Anees Ahmed Khalil 2, Faryal Shaukat 1 and Yuanda Song 1,\***

Received: 16 July 2019; Accepted: 28 July 2019; Published: 1 August 2019

**Abstract:** In the recent era, bioactive compounds from plants have received great attention because of their vital health-related activities, such as antimicrobial activity, antioxidant activity, anticoagulant activity, anti-diabetic activity, UV protection, antiviral activity, hypoglycemia, etc. Previous studies have already shown that polysaccharides found in plants are not likely to be toxic. Based on these inspirational comments, most research focused on the isolation, identification, and bioactivities of polysaccharides. A large number of biologically active polysaccharides have been isolated with varying structural and biological activities. In this review, a comprehensive summary is provided of the recent developments in the physical and chemical properties as well as biological activities of polysaccharides from a number of important natural sources, such as wheat bran, orange peel, barely, fungi, algae, lichen, etc. This review also focused on biomedical applications of polysaccharides. The contents presented in this review will be useful as a reference for future research as well as for the extraction and application of these bioactive polysaccharides as a therapeutic agent.

**Keywords:** bioactive polysaccharides; extraction; biomedical applications

#### **1. Introduction**

Polysaccharides are considered as vital bio-macromolecules for all living organisms, which are structurally comprised of homo or hetero monosaccharides and uronic acids connected with glycosidic linkages [1–3]. They are predominantly found in various parts of plants, animals, fungi, bacteria, and seaweed, and play a critical role in numerous physiological functions of life [4]. Polysaccharides along with lipids, proteins, and polynucleotides are classified as the most pivotal four macromolecules in life sciences. Bioactive polysaccharides are known as polysaccharides produced from living organisms, and/or are functionalized from sugar-based materials possessing biological effects on organisms. Moreover, during the last decades, bioactive polysaccharides have been investigated as therapeutic agents against many chronic diseases due to their biodegradability, non-toxic nature, and biocompatibility [5]. Studies have shown that polysaccharides possess a wide range of pharmacological perspectives such as antioxidative, antitumor, antimicrobial, anti-obesity, hypolipidemic, antidiabetic, and hepato-protective properties [6–8]. They have been investigated extensively for the development of novel products in the field of cosmetics, food, medicine, petrochemicals, and paper [3,9,10]. Particularly, in the medicinal industry, polysaccharides are mostly used as pharmaceuticals and medical biomaterials (hypoglycemic, anti-osteoarthritic, and anticancer products) to curtail the effect of respective metabolic syndromes [9,11].

The potentiality of bioactive polysaccharides is strongly influenced depending upon their configuration and chemical structure. Nevertheless, the macromolecular configurations of plant cellular polysaccharides, particularly hetero polysaccharides (hemicelluloses), are very complex owing to the occurrence of various monosaccharides acting as isobaric stereoisomers [12,13]. Additionally, polysaccharides present in plants, microorganisms (bacteria, fungi, and yeasts), animals, and algae are chemically and/or physically bound with various other biomolecules like lignin, proteins, lipids, polynucleotides, and a few minerals [14]. Hence, to understand the importance of bioactive polysaccharides in the domain of life sciences resulted in the multi-disciplinary collaboration of scientists from the fields of microbiology, phytology, glycol-biology, nutrition, food sciences, and glycol-medicine [5]. Polysaccharides both in the simple and complex glycol-conjugated form are renowned for various bioactive functions, for instance, antivirus, antioxidant, antimicrobial, anticancer, antidiabetic, reno-protective, immunomodulatory, and anti-stress perspectives.

#### **2. Sources of Bioactive Polysaccharides**

Bioactive polysaccharides are categorized broadly depending upon their sources, structure, applications, solubility, and chemical composition. On the basis of chemical composition, they are characterized as homopolysaccharides (homoglycans) and heteropolysaccharides (heteroglycans). Homoglycans comprise of a single type of monosaccharide, such as glycogen and cellulose which consists of glucose molecules, whereas heteroglycans are made up of a different type of monosaccharides, for example, heparin and chondroitin sulfate (CS) [15].

According to the glycosides linked on to the glycan, polysaccharides can also be classified as proteoglycans and glycoproteins, glycolipids, and glycoconjugates [16,17]. According to grouping based on origins, bioactive polysaccharides are usually classified as those derived from animals (chondroitin sulfate, heparin, and hyaluronan), plants (pectin, inulin, Ginseng polysaccharides, xylans, and arabinans), bacteria (exopolysaccharides, capsular polysaccharides, and peptidoglycans), lichen, fungi, and algae. In the section below, bioactive properties associated with polysaccharides are reviewed depending upon their natural sources in order to understand their functionality.

#### *2.1. Bioactive Polysaccharides in Dietary Fibers*

The FAO (Food and Agriculture Organization) defined dietary fibers as a variety of indigestible plant polysaccharides including pectins, cellulose, gums, hemicelluloses, oligosaccharides, and various lignified compounds. Dietary fibers are the type of polysaccharides that are biologically active in their innate form and/or after enzymatic and chemical treatments. For instance, celluloses and hemicelluloses directly stimulate the bowel movement, whereas inulin is fermented by microflora with the intent to prevent various gastrointestinal disorders [18]. Dietary fibers play a vital role in prevention from various diseases especially hyperlipidemia, cardiovascular complications, and obesity. Gums, inulin, and pectin are capable of slowing down the passage of food in the digestive tract, reducing serum cholesterol concentrations, hindering the absorption of sugar from food to bloodstream and, therefore, avoiding sudden hyperglycemic conditions post food ingestion.

Among dietary fibers, lignin, cellulose, and hemicellulose are classified as insoluble fibers which help in the stimulation of the bowel movement, speeds up the removal of waste via the digestive tract, and helps in prevention of hemorrhoids, constipation, and diverticulosis [19,20]. Results of various epidemiological investigations and clinical trials have suggested that consumption of moderate or higher levels of dietary fiber efficiently reduces the risk of numerous metabolic syndromes like diabetes [21], strokes [22], hypertension [23,24], hyperlipidemia/hypercholesterolemia, [25,26], obesity, and cancer [27]. Usually, ingestion of dietary fiber has health modulating benefits, like increased stool bulk, reduced intestinal transit time, a decline in levels of total cholesterol (TC) and postprandial blood glucose, and/or insulin contents [27–29]. Keeping in view the significance of dietary fibers with special reference to human health and their associated mechanism of actions, these are considered as an abundant source of bioactive polysaccharides. Table 1 summarizes the types, names, sources, composition, and physiological effects of dietary fiber polysaccharides from various studies and trials.

#### *2.2. Bioactive Polysaccharides in Herbs*

Herbs have attained a significant position in traditional medicines (Indian Ayurveda, ancient Chinese medicine, phytomedicine in western countries, and Japanese Kamp medicine) of numerous countries owing to their associated curing benefits against numerous diseases. Outcomes of recent pharmacological studies have illuminated that the chief component of herbal medicines usually comprises of tannins, polysaccharides, high molar mass proteins, and low molar mass constituents like terpenoids (quassinoids, sesquiterpenes, and *Rabdosia* diterpenes), saponins, alkaloids (protoberberine alkaloid, phenanthridine, etc.), and flavonoids (*Scutellaria* flavones) [30]. Among the above-mentioned compounds in herbal medicines, polysaccharides are believed to be main bioactive compounds responsible for various pharmacological potentials like antitumor, antioxidant, hepato-protective, antiviral, radio-protective, immuno-stimulatory, and anti-fatigue activities [31–35]. Innate polysaccharides present in numerous herbs are known for stimulation of the human immune system, inhibition of viral replication, scavenging free radicals, and inhibition of lipid peroxidation [33,36,37]. Recent advancements regarding the application of polysaccharides present in herbal medicines for prevention and cure of diseases are documented in Table 1.

#### *2.3. Bioactive Polysaccharides in Algae and Lichens*

Polysaccharides present in lichens and algae have been of great interest to the food scientists owing to their associated exceptional physical properties (stabilizing, gelling, and thickening capability) and biological potentials (immunomodulating, antiviral, anticoagulant, antitumor, antioxidative, anti-inflammatory, and anti-thrombotic activity) [48,49]. Basically, sulfated polysaccharides are the main group of the most attractive and interesting constituents in marine algae i.e., laminarans and fucoidans in the Phaeophyceae (brown algae), carrageenans in the Rhodophyceae (red algae), and ulvans in the Chlorophyceae (green algae) [40]. One of the most vital properties linked with sulfated polysaccharides is their anticoagulant characteristics. For instance, carrageenan (sulfated galactans) isolated from red algae and sulfated fucoidans extracted from brown algae are known to possess excellent anti-coagulant properties. Sulfated polysaccharides identified in algae have been stated to own equal or even stronger activities than those linked with heparin [32]. Various other biological properties of sulfated polysaccharides present in algae have been intensively investigated.

Experimentally, fucoidan has been reported to possess maximum antioxidative properties followed by alginate and laminaran, therefore protecting human health from the damage of ROS (reactive oxygen species). Likewise, the anticancer and anti-tumor characteristics of algae-based sulfated polysaccharides are found to be due to their antioxidative and free radical scavenging properties [50]. Moreover, inhibition of HIV and herpes viruses in cells owing to anti-viral potential of sulfated rhamnogalactans, carrageenans, and fucoidans have also been scientifically proven [40]. Immuno-modulating characteristics associated with algae-based sulfated polysaccharides were demonstrated by increasing the secreting and phagocytic activities of macrophages [51].

Polysaccharides derived from lichens (composite organism formed due to a symbiotic partnership among alga and fungus) are principally linear or rarely substituted α-glucans and/or β-glucans. This type of glucans isolated from lichens is reported to have a wide range of bioactive functions such as immunomodulatory perspectives, anti-tumor potential, and anti-viral characteristics [49,52,53]. The immuno-modulating activity of polysaccharides (β-glucans) derived from lichenin and lichens are linked to stimulation of a variety of immune responses like production of NO (nitric oxide) and ROS (reactive oxygen species) and release of cytokines and arachidonic acid metabolites [51]. Lichenin, also known as lichenan, consists of a galactoglucomannan (water-soluble hemicellulose) structure that is responsible for anti-thrombotic and anti-coagulant properties [54]. Bioactive properties related to sulfated polysaccharides from lichens and algae are highly dependent on their structural properties, for instance, sulfate concentration and distribution of sulfate groups on the main chain, stereochemistry, and molar masses. Hence, there is a need for the modification of innate sulfate polysaccharides to attain biologically active polysaccharides having desired molecular size and functionality for bioactive further application [40,55]. An overview of some recent advancements in this context are depicted in Table 2.

#### *2.4. Bioactive Polysaccharides in Fungi*

A wide range of bioactive saccharides is present in fungi ranging from monosaccharides to polysaccharides. They can be summarized as the intracellular or extracellular polysaccharides depending upon their presence in the fungi [56]. The bioactive polysaccharides derived from basidiomycetes are reported to have antitumor and anticancer activities and can prevent breast cancer in post-menopausal women. The compound which can inhibit tumors was first isolated from the basidiomycete *Boletus edulis* in 1957. In traditional medicine, several basidiomycete mushrooms are used as an immunosuppressant to treat cancer and even for treating AIDS [57]. Polysaccharopeptide (PSP) isolated from *Ganoderma lucidum* induces the inhibition of oncogenes and kill the tumor cells directly. Similarly, the bioactive polysaccharides schizophyllan, lentinan, and polysaccharide-K isolated from *Grifola frondosa* and *Trametes versicolor*, respectively, improve the immune function of the host and showed a synergistic effect with chemotherapy [58]. Just like mushrooms, another source of bioactive polysaccharides is *Cordyceps militaris*, and they possess a wide range of structurally different bioactive polysaccharides. Water-soluble polysaccharides from *C. militaris* contain a (1 → 4) galactose linkage and (1 → 3,6)-mannose linkage and branching usually arising from (1 → 4)-linked glucose. These polysaccharides were reported for their numerous biological activities including anti-tumor, immunomodulatory, antioxidant, and anti-inflammatory activities [56,59].

#### *2.5. Bioactive Polysaccharides from Bacteria*

Bacteria are a rich source of biologically active polysaccharides. On the base of structural properties, bacterial polysaccharides can be defined into five major classes: Exopolysaccharides (EPSs), capsular polysaccharides (CPSs), lipopolysaccharides (LPSs), peptidoglycans, and teichoic acids [60].

A lot of research has been done on hyperbranched bacterial polysaccharides (HBPSs). For example, highly branched dextran with a backbone of 75% (α-(1→6)-D-Glcp) with 19% of α-(1→3) and a few α-(1→2) branching [61], was separated from *Leuconostoc citreum* B-2 and studied extensively, and showed a water-holding capacity of up to 450% and an 80% water solubility index, consequently having potential applications in cosmetic, food, and pharmaceutical industries [62,63]. From an industrial point of view, EPSs producing Gram-negative prokaryotes (cyanobacteria) are important because of their ability to produce novel molecules. These EPSs usually linger as a sheath/capsule with the cells and when they are liberated from the cells they are termed as released polysaccharides. Some bacteria from the genus cyanobacterium, namely *Anabaena, Aphanocapsa, Cyanothece, Phormidium, Synechocystis,* and *Nostoc* are useful to produce sulfated EPSs, which also contain uronic acids [64]. Upon fermentation in the laboratory, polysaccharide yield from 0.5–4 g/L can be obtained from three known genera of marine bacteria, namely *Alteromonas* sp., *Pseudoalteromonas* sp. and *Vibrio* sp. [65]. Exopolysaccharide HE 800 is secreted by *Vibrio diabolicus* and it contains an equal amount of hexosamine and glucuronic acid, which can be depolymerized, N-deacetylated, and after chemical sulfurylation produce new derivatives of HE800. These derivatives are referred to as DRS HE800 and are structurally very close to heparan-sulfate [66]. Moreover, acidic and highly branched heteropolysaccharide (EPS GY785) is produced by the bacterium *Alteromonas infernus*. Non-saccharide units of EPS GY785 are composed of uronic acid and sugar (glucose and galactose) [67].

EPSs derived in the laboratory showed widespread biological activities like being an antioxidant, having cholesterol-lowering and inflammation-regulating properties, as well as anti-tumor, anticoagulant, and antivirus activities [62,68]. Moreover, bacterial polysaccharides are used in Protein Glycan Coupling Technology (PGCT) to produce glycoconjugate vaccines against Gram-positive and Gram-negative bacteria [69].

#### *2.6. Bioactive Polysaccharides in Wood*

Bioactive polysaccharides present in wood primarily consists of celluloses and some primary groups of hemicelluloses (glucans, arabinans, xylans, glucomannans, and galactans) [70,71]. Pectins and galacto-glucomannans derived from wood are stated to have radical scavenging properties and immune-stimulating activities [72,73]. Xylo-oligosaccharides (xylans) derived from dietary fibers, hard and/or softwood have been reported to be used efficiently as prebiotics by nutraceutical and medicinal industries [74]. Similarly, pentosane polysulfate—a xylan derivative being extracted from beech-wood—is also known as a biomedical agent for the treatment of interstitial cystitis, a bladder pain syndrome [75]. While most polysaccharides do not reveal biological properties unless they are subjected to some modification; derivatives of cellulose-like hydroxypropyl-cellulose (HPC), hydroxypropyl-methylcellulose (HPMC), hydroxyethyl-cellulose (HEC), and methyl-cellulose have evidently proven their functional roles in numerous fields (medicinal, cosmetics, food, and pharmaceutical) [76].

#### *2.7. Bioactive Polysaccharides from other Sources*

Sulfated glycosaminoglycans (GAGs) like keratin sulfate (KS), heparan sulfate (HS), dermatan sulfate (DS), and chondroitin sulfate (CS) belongs to a class known as animal-derived bioactive polysaccharides. Heparan sulfate comprises of repeated units of N-acetylated and sulfated disaccharides (glucosamine and glucuronic/iduronic acid) units [77–79]. Evidently, heparin is known to be the most effectual clinical anti-coagulant used in medical sciences. The anti-coagulation potential of heparin is mainly due to its particular structural arrangement, in which specifically the binding site of anti-thrombin III (ATIII, a non-vitamin K-dependent protease) is of central importance in the prevention of fibrin clot formation which is generated owing to the enzymatic activity of thrombin [80]. Better knowledge regarding the interlinkage among structural properties and activity of heparin creates an opportunity for the development of new drugs having more specificity and improved anti-coagulating properties. Furthermore, this structural-activity connection is also helpful in exploring various biomedical applications like anti-viral, anti-inflammatory, anti-cancer, and wound healing properties [78,79,81,82]. DS and CS chains comprise of sulfated N-acetylgalactosamine and iduronic acid (in case of DS)/glucuronic acid (in case of CS) disaccharide repeating units [83,84]. Both of these are functionally characterized as bioactive polysaccharides owing to their presence as a vital molecule in the extracellular matrix of the brain, which helps in regulation of cell migration, adhesion, and proliferation while healing of wounds, as well as proper signaling of growth factor in the skeleton [85–88].

On the other hand, HA (hyaluronic acid) primarily isolated form animal tissues comprise of the linear structure of non-sulfated glycosaminoglycan molecules with repeated units of β-(1-3)-*N*-acetyl-d-glucosamine, and β-(1-4)-d-glucuronic acid. It is also a vital constituent of the brain's extracellular matrix, which promotes mediation of cellular signaling, helps in healing of wounds, and morphogenesis. Owing to these properties, HA and their associated derivatives are predominantly being applied by the medical industry in eye surgery, viscosupplementation, and as a biomedical tool for drug delivery [89–91]. Chitin is known to be the most abundant polysaccharide present in the exoskeleton of shrimps and crabs, the cell walls of yeast and fungi, and the cuticles of insects. It is by far the second most copious polymer after cellulose and comprises of a random distribution of β-(1-4)-linked *N*-acetyl-glucosamine and *N*-glucosamine [92,93]. Industrially, chitosan is characterized as a vital derivative of chitin and is practically obtained due to partial deacetylation of chitin either by the action of enzyme chitin deacetylase or under alkaline conditions [94]. Bioactive properties (anti-cancer, anti-tumor, anti-bacterial, anti-inflammatory, etc.) associated with oligomers of chitosan and chitin, yielded owing to partial acidic hydrolysis, have earlier been documented [95].

#### **3. Extraction and Quantification of Bioactive Polysaccharides**

In general, bioactive potentials of naturally occurring polysaccharides are greatly dependent upon their molar mass and level and distribution of groups/side chains on the backbone. Therefore, isolation of polysaccharides from complex cellular plant matrices while keeping their bioactivity intact is of great significance. During the last decade, various innovative green extraction techniques (microwave-assisted, ultra-sonication, supercritical fluid extraction, and hot water extraction) are in practice for isolation of bioactive polysaccharides. These techniques have acquired great attention of scientists and researchers mainly due to their increased extraction rates, cost-effective nature, enviro-friendly characteristics, and structure preservative potentials [73,96,97].

Numerous scientific investigations have been implemented for the extraction and isolation of bioactive polysaccharides. The type of method adopted defines the physicochemical properties and antioxidant potential of isolated polysaccharides. For this purpose, a group of scientists compared the structural and antioxidant properties of bioactive polysaccharides extracted from Barrenwort (*Epimedium acuminatum*) through various extraction techniques (microwave-assisted, enzymatic, hot water extraction, and ultrasound-assisted extraction). They were of the view that bioactive polysaccharides isolated from hot water extraction had the highest antioxidant properties as compared to those extracted from other techniques, while their physicochemical properties were the same [98]. The hot water extraction method used in combination with various latest techniques (enzymatic pre-treatment, microwave, and ultrasound-assisted) are useful in increasing the yield and extraction productivity of polysaccharides. Likewise, enzymatic pre-treatment of raw material prior to

extraction resulted in reduced extraction time, minimized the use of extraction solvents, preserved the bioactivities of the polysaccharides, and was energy efficient as compared to non-enzymatic pre-treated techniques [99]. In recent times, various ionic liquids have been formulated which aids in extraction of polysaccharides in a shorter time and at lower temperatures [100].

**Table 2.** Extraction techniques with reference to specific polysaccharides.

In addition, this purification of polysaccharides from the crude extract is really of great importance as the linkage among structure and safety of products formed for food, pharmaceutical, and biomedical application depends on this. Purification could be achieved by using various techniques (gel filtration, ion exchange and affinity chromatography, ethanolic precipitation, and fractional precipitation), individually or in combination [32].

#### **4. Biomedical Applications**

Polysaccharides and their derived compounds are medicinally more preferred as compared to synthetic polymers owing to their biodegradability, non-toxic nature, biocompatibility, and low processing expenses. Mentioned benefits related to polysaccharides isolated from natural sources make them a valuable ingredient in the fields of pharmaceuticals, nutraceuticals, food, and cosmetic industries. At the present time, polysaccharides are been used in healthcare and disease control, while various novel areas have also been discovered like in cancer diagnosis, inhibition, and treatment; in drug delivery; in anti-bacterial and anti-viral perspectives; and in tissue engineering [92,119]. Therefore, this segment highlights the use of bioactive polysaccharides against various metabolic syndromes and in the above-mentioned novel areas.

#### *4.1. Anti-Microbial and Antiviral*

Various clinical investigations have authenticated that oral administration of pectin to infants and children significantly reduced diarrhea and other intestinal infections. This may be because of the decreased concentration of pathogenic bacteria like *Citrobacter*, *Salmonella*, *Enterobacter*, *Shigella*, *Proteus*, and *Klebsiella* [120]. A linear relationship has been documented among the concentration of probiotics and intestinal health [28].

The bioactive potential of fucoidans—a sulphated polysaccharide derived from marine brown seaweeds—have demonstrated noteworthy anti-viral potential against the cytomegalovirus, HIV, and HSV (herpes simplex virus) [121]. Additionally, few other seaweed-extracted polysaccharides like sulphated rhamnogalactans, carrageenans, and fucoidans have shown an inhibitory effect on viruses (HSV and HIV). Fucoidan comprises of a large quantity of L-fucose and sulphate groups along with fractions of galaturonic acid, xylose, mannose, and galactose. *Undaria pinnatifida* (marine brown alga) contains fucoidans and have been used in bone health supplementation mainly due to stimulation of osteoblastic cell differentiation. This sulphated polysaccharide has also been known to possess preventing action on UV-B-induced matrix metalloproteinase-1 (MMP-1) expression by inhibiting the ERK (extra-cellular signal regulated kinases) pathways. Therefore, it could be utilized as a functional ingredient in dermal ointments to prevent from skin photo-aging [40].

Some of the other fractions of algae have properties of virucidal and enzyme inhibitory activity inhibiting the formation of the syncytium. Besides, the sulfate group present is necessary for the anti-HIV activity and potency increases with the degree of sulfation.

#### *4.2. Anti-Tumor*/*Cancer*

Numerous scientists have explored dietary fibers as possessing potent anti-cancer properties. Amongst all, pectin has been investigated to reduce cancerous cell migration and tumor growth in a rat model that were administrated with modified citrus pectin [122]. This may be due to binding of pectin to galectin-3, which results in inhibitory action on some of its functional activities [123]. Anti-tumor mode of actions associated with dietary pectin are related to their immune-potentiation, probiotic properties, tumor growth inhibition, anti-mutagenic potential, and regulatory action of transformation-related oncogenes [124,125]. Anti-tumor mechanisms associated with pectin could be due to cellular immunological potential [126].

According to a study, ginseng polysaccharides were found to have a stimulating effect on DCs (dendritic cells) causing an elevated formation of IFN-g (interferon-g) [127]. It has also been documented that acidic ginseng polysaccharides (GPs) enhanced the production of cytotoxic cells against tumors and promoted macrophages for the production of Th1 and Th2 (helper type 1 and 2) cytokines [128,129]. Depending upon disease environment or timing of treatments, ginseng polysaccharides extracted from *Panax ginseng* demonstrated immuno-modulating perspectives mainly in an immunosuppressing or immuno-stimulating manner [130]. Acidic GP also revealed modulating action on the concentrations of antioxidative enzymes like GPx (glutathione peroxidase) and SOD (superoxide dismutase) probably due to induction of regulating cytokines [131,132]. Likewise, Lemmon et al. [132] found that the immuno-stimulating potential of acidic GPs isolated from American ginseng (*Panax quinquefolius*) was actually mediated by polysaccharides having molecular weight more than 100 kDa [34].

Furthermore, scientists have proven the fact that heparin administration may also have a beneficial impact on cancer and inflammation. Anti-cancerous, anti-inflammatory, and anti-tumor properties associated with heparin and its low molecular weight species are owing to the pathological functions of heparan sulfate (HS) chains of proteoglycan structure (HSPGs). Outcomes of an investigation validated that heparin transfers GRs (growth factors) stored by HS chains of HSPGs in the ECM (extracellular matrix) and on cell surfaces. Full-size heparin has potent pro-angiogenic properties as it increases the production of ternary complexes of heparin bound FGF2 and VEGF with GF receptors [45].

#### *4.3. Anti-Obesity and Hypocholesterolemia*

Numerous trials have shown a direct relationship between consumption of dietary fiber, rich diet/dietary fiber supplementation, and weight loss [133–137]. According to a meta-analysis comprising of 22 clinical trials, it was documented that a 12 g increase in the content of daily fiber intake resulted in a 10% decrease in energy intake along with a 1900 g decline in body weight [138]. More precisely, the administration of glucomannan (1.24 g/day) along with energy-restricted diet for five consecutive weeks caused a significant decrease in body weight as compared to the placebo group [139].

In a clinical trial on healthy volunteers, a drink containing oat β-glucan (10.5 g/400 g and 2.5 and 5 g/300 g) enhanced fullness sensation as compared to fiber-free drink [140,141]. Likewise, in healthy adolescents subjected to biscuits enriched with barley β-glucan (5.2%) helped in suppressing appetite ratings as compared to control biscuits [142]. Similarly, administration of bread formulated by barley β-glucan (3%) to volunteers resulted in decrease of hunger and increased satiety and fullness. This also resulted in a noteworthy decrease in energy intake at successive lunches [143]. On the other hand, a bar prepared from barley β-glucan (1.2 g) subjected to healthy volunteers did not change scores for energy intake and appetite scores as compared to control bars [144]. Effects of β-glucan on satiety depends upon the concentration, molecular weight (31–3100 kDa), solubility, and food carrying it [38].

Furthermore, a group of scientists investigated the hypocholesterolemic perspectives of a dietary supplement comprising equal content of konjac glucomannan (KGM) and chitosan [145]. The concentration of serum total cholesterol and low-density lipoprotein cholesterol (LDL-c) significantly reduced at the end of the trial (28th day). Fecal excretion of bile acids and neutral sterol were observed more at the commencement of the study as compared to the initiation of the study. Similarly, Chen et al. [146] investigated the impact of KGM supplementation (3.6 g/day) on levels of glucose and lipid biomarkers in hypercholesterolemic type-2 diabetic patients. Twenty-two diabetic patients having increased serum cholesterol content were selected for this study. As compared to the placebo group, KGM supplemented group showed decreased levels of LDL-c (20.7%), fasting glucose (23.2%), serum cholesterol (11.1%), and Apo-B (12.9%). Fecal bile acid and neutral sterol content were elevated significantly by 75.4% and 18.0%, respectively. Results of all the mentioned trials revealed that KGM supplementation could assist in the treatment of hypercholesterolemic diabetic patients [44].

#### *4.4. Anti-Diabetic*

Scientific evidences have shown that β-glucan can contribute to control glycemic responses. Numerous factors are found to affect such interactions like the nature of the food, concentration, and molecular weight of β-glucan. Among all these, the dose of β-glucan is considered to be the most important factor in regulating the impact of fiber on glycemic responses. As compared to other fibers,

a small dose of β-glucan is sufficient to reduce the insulin and postprandial glucose responses in type 2 diabetic [147,148], healthy [149,150], and hyperlipidemic subjects [151]. Studies have revealed that consumption of breakfasts comprising of 4, 6, and 8.6 g of β-glucan momentously reduced the mean concentration of serum insulin and glucose as compared to control non-insulin-dependent diabetic mellitus subjects [147]. The content of exogenous glucose was noticed as 18% less in a polenta meal containing oat β-glucan (5 g) as compared to a control polenta meal without oat β-glucan-subjected individuals [152]. Likewise, consumption of a meal consisting of 13C-labelled glucose and β-glucan (8.9 g), for a period of three days, reduced (21%) the levels of exogenous 13C-glucose in plasma as compared to control meal having no β-glucan [38,153].

#### *4.5. Gastro-Protective*

An experimental trial conducted by means of two diverse types of resistant starches (one a high amylose granular resistant corn starch and the other was high amylose non-granular, dispersed, and retrograded resistant corn starch) to evaluate the influence on blood lipid concentration, fecal SCFA and bulking, and glycemic indexes. This study also comprised of supplements containing low fiber control and high fiber control. Outcomes of this trial revealed that high fiber control (wheat bran) and both resistance starches subjected groups showed an elevation in the fecal bulk as compared to the low fiber control group. Likewise, the average ratio of fecal SCFAs and butyrate had progressive effects on colon health. Xanthan gum may also be used in milk as a prebiotic for lactic acid bacteria. Similar trials regarding prebiotics have demonstrated protective implications on the sustainability of cultures under the presence of bile salts and refrigeration and low pH conditions. According to a study, guar gum has the capability to change lipoprotein and postprandial lipid compositions. Supplementation of guar gum has an influence on lipoprotein composition, lipemia, and postprandial glycaemia [19].

Chen et al. [154] explored the effect of KGM supplementation on the gastrointestinal response in volunteer subjects. They were of the view that KGM supplementation significantly elevated the dry and wet stool weight and defecation frequency to 21.7%, 30.2%, and 27.0%, correspondingly. The improved dry fecal mass may be due to the existence of plant soluble materials. Nevertheless, the bacterial biomass of total bacteria, bifido-bacteria, and lactobacilli increased in fecal mass in KGM supplemented groups. Furthermore, reduction in fecal pH and elevation in fecal short chain fatty acids (SCFAs) resulted in increased colonic fermentation owing to KGM supplementation [44].

#### *4.6. Immune Modulatory*

Ginseng polysaccharides (GPs) have not only been known to possess immune-stimulating perspectives but also are found to suppress the proinflammatory responses. According to a recent study, novel neutral polysaccharide (PPQN) derived from an American ginseng root was documented to have a suppressing effect against inflammation. This activity was reported due to the inhibitory effect of isolated polysaccharide on inflammatory-related mediators such as cytokines (IL-1b, IL-6, TNF-a) and NO (nitric oxide) in comparison with LPS (lipopolysaccharide) treatment. Owing to this mode of action, novel neutral polysaccharide isolated from an American ginseng root could be used in modulating numerous inflammatory-related health implications (tumor, cancer, etc.) [155]. Similarly, another study reported the inhibitory influence of ginseng polysaccharides on immunological responses noticed in collagen-induced arthritic subject [156]. *P. quinquefolius* (American ginseng) is extensively used for the preparation of numerous herbal products. Extracts of *P. quinquefolius* were found to suppress the immune-inflammatory response, reduced the activity of neutrophils, induced the formation of cytokines in the spleen, and elevated the production of splenic-B lymphocytes and bone marrow [157–160].

*Platycodon grandifloras* is an herbaceous plant which is used as folk medicines since ancient times to curb various diseases like asthma, bronchitis, and pulmonary tuberculosis. Proximate composition of *P. grandifloras* reveals that it is a rich source of carbohydrates (90%), protein (2.4%), ash (1.5%), and fat (0.1%). Polysaccharides extracted from roots of *P. grandifloras* have been reported to possess

antidiabetic, hypolipidemic and hypocholesterolemic properties [161]. Furthermore, the inulin-type polysaccharides isolated from *P. grandifloras* (PGs) roots validated the immune-modulating impact on macrophages and B-cells, but had no effect on T-cells [162].

#### *4.7. Anti-Inflammatory*

*Astragalus* polysaccharides (APS) are known to possess anti-inflammatory effects on cytokines of CD4+ Th (T-helper) cells. In in-vitro antidiabetic models, an *Astragalus* polysaccharide has potentiated the lowering effect on the expression of T-helper 1 (Th1) and regulated the imbalance of Th1 and Th2. APS has reported to significantly enhance the gene expression of peroxisome-proliferator-activated receptor gamma (PPAR-γ) in a concentration-time dependent manner [163] and stimulated superoxide dismutase (SOD) anti-oxidative mechanism in type-1 diabetes mellitus (DM) models [164,165]. Moreover, APS reduced the expression of iNOS (inducible nitric oxide synthase) [122]. These inflammatory markers (NO, PPAR-γ, SOD, and iNOS) amongst diverse roles also perform numerous functions in regulating and stimulating inflammatory response [42].

Water-soluble sulfated polysaccharides (WSSPs) isolated from marine algae are also classified as anti-inflammatory compounds. On the other hand, very few pieces of evidence are present regarding anti-inflammatory perspectives of seaweed based sulfated polysaccharides. In vitro and in vivo studies have revealed that *Gracilaria verrucose-* and *Porphyra yezoensis*-derived sulfated polysaccharides stimulated the respiratory burst and phagocytosis in experimented mouse macrophages [40]. Orally administrated chondroitin sulfate (CS) isolated from cartilage of Skate (*Raja kenojei*) affected arthritic conditions in a dose-dependent manner in chondroitin sulfate-treated groups. Pre- and post-treated groups that were subjected to CS (1000 mg kg−1) revealed momentously decreased clinical scores as compared to vehicle treated groups. CS administration decreased the infiltration of inflammatory cells and prohibited from paw and knee joint destruction. Moreover, the results of RT-PCR showed that CS ingestion significantly repressed the expression of IL-1b (interleukin-1b), IFN-c (interferon-c), and TNF-α as compared to vehicle administrated group. The CS-treated group reduced the formation of rheumatoid arthritis responses (IgG and IgM) in collagen-induced arthritic mice (CIA) model. Outcomes of this study authenticate the shielding potential of chondroitin sulfate in CIA mice mainly due to the inhibitory effect of pro-inflammatory cytokines formation [43].

#### *4.8. Neuro-Protective*

*Acanthopanax senticosus* derived polysaccharides comprised of uronic acid (22.5%), proteins (18.7%), and carbohydrates (58.3%). It could be established that *Acanthopanax*-based polysaccharides may not only help in improving symptoms regarding nervous defects but also reduced the infarct volume and water content of the brain in rats having cerebral ischemia–reperfusion injury. Additionally, polysaccharides isolated from *A. senticosus* elevated SOD, IL-10, and GSH-Px concentration and reduces the levels of TNF-α, IL-1, and MDA in brains tissues of experimented rats. Conclusively, bioactive polysaccharides extracted from *A. senticosus* protected brain damage due to antioxidative potential and inhibitory action on stimulation of inflammatory cytokines [47].

#### *4.9. Anti-Oxidant*

Bioactive acidic polysaccharides extracted from *Polygonum multiflorum* showed significant antioxidative properties (hydroxyl peroxide, superoxide anion radical, and hydroxyl radical), protein glycation and lipid oxidation. In addition to this, the intraperitoneal (i.p.) administration of *P. multiflorium*-based polysaccharides may increase the serum concentration of antioxidative characteristics in cyclophosphamide-induced anemic mice. Results of this study validate the use of *P. multiflorium* as a novel antioxidant tool to prevent oxidation [41]. Sulfated polysaccharides not only act as dietary fiber but also act as a natural antioxidant agent. They are responsible for the antioxidant properties possessed by marine algae. Various studies have recognized the use of numerous classes of SPs (alginic acid, Fucoidan, and laminaran) as potent antioxidative agents. Antioxidative potential of SPs has classified by multiple in-vitro methods such as DPPH, FRAP, NO, ABTS radical scavenging, superoxide radical scavenging assay, and the hydroxyl radical scavenging assay. Additionally, Xue et al. [166] stated that many marine-based sulfated polysaccharides have shown antioxidant potential in organic solvents and a phosphatidylcholine-liposomal suspension [40].

#### *4.10. Tissue Engineering*

Application of bioactive polysaccharides and their derivatives in the field of tissue engineering (cell differentiation, cell adhesion, cell remodeling, cell proliferation, and cell responsive degradation) has opened new horizons in medical research, and therefore impelled the researchers to regenerate new tissues and define the structure of cellular growth [92]. Various bioactive polysaccharides including starch, chitosan, chondroitin sulfate, alginate, cellulose, chitin, hyaluronic acid, and their derivatives are being used as biomaterials in applications for tissue engineering [167]. Application of these bioactive polysaccharides as scaffolds in tissue engineering are required to accomplish some requirements such as non-toxicity, biodegradability having controlled the rate of degradation, biocompatibility, structural integrity, and suitable porosity [92].

Chitosan and chitin have all the required potential to act as scaffolds for tissue engineering mainly due to their mechanical strength, degradability, and immunogenicity. Hence, for tissue engineering they are being developed as 3D-hydrogels, free standing films, porous sponges, and fibrous scaffolds, inside which for in-vitro/in-vivo cultures the most suitable cell types are needed [168]. Designing of 3D-chitin/chitosan-based hydrogels and sponge scaffolds, and 2D-scaffolds for the purpose of cartilage and tendon regenerations, for encapsulation of stem cells ensuring their therapeutic application, and for utilizing these as a tool for regenerative medicine have been reported in numerous researches [169,170]. Furthermore, for bone regeneration purpose, the tissue engineering industry has formulated combinations of chitosan and hydroxyapatite and grafted chitosan and carbon nanotubes [171]. Along with this, numerous other bioactive polysaccharides like cellulose, hyaluronic acid, and starch have also been studied in detail to validate their use as a biomaterial for skin, bone, and cartilage tissue engineering [95].

#### *4.11. Wound Healing and Wound Dressing*

Numerous bioactive polysaccharides (alginate, chitin, hyaluronan, chitosan, and cellulose) are used for the preparation of wound healing materials owing to their intrinsic bio-compatible, less toxic, and pharmaceutical activities [172,173]. For instance, hyaluronan is a vital extracellular component possessing distinctive viscoelastic, hygroscopic, and rheological characteristics are well known for its tissue repairing properties owing to their physicochemical potentials and specific interaction with cells and extracellular matrices. It is documented earlier that hyaluronan has a multidimensional role regarding the repairing process of cell or wound healing specifically inflammation, granulation, formation of tissues, re-epithelialization, and remodeling. Various hyaluronan-derived products like esterified, cross-linked, or chemically modified products are medicinally used for wound healing and tissue repairing purposes [174]. While designing bioactive material for tissue engineering their wound healing properties is of great interest.

Naturally available wound dressing films either prepared by encapsulation or simply dispersion of the sodium alginate matrix in essential oils from cinnamon, lemon, tea, lemongrass, lavender, elicriso italic, peppermint, chamomile blue, and eucalyptus have demonstrated exceptional anti-fungal and anti-microbial activities, and therefore their application in disposable dressings for wounds could also be found [175]. Development of wound dressings obtained from cross-linkage between chitosan/silk fibroin blending membranes and di-aldehyde alginate have found to enhance cellular proliferative properties, suggesting their applications as wound healing agents [176]. Preparation of freestanding sodium alginate films or Ca2þ cross-linked alginate beads was achieved by mixing aqueous dispersions of PVPI (povidone iodine) and Na-Alg. These films/beads showed anti-fungal/anti-bacterial properties along with control release of povidone iodine into wounds as these products came into

direct contact with the moist environment [175]. These applications validate the use of these products therapeutically in wound dressings. Some innovative wound dressings were prepared for external treatment of wounds by in situ injection of nanocomposite hydrogels that actually comprised of oxidized alginate, curcumin, and N, O-carboxymethyl chitosan. Results of various in vitro, in vivo, and histological investigations have proven the use of nanocurcumin, N, O-carboxymethyl chitosan, and oxidized alginate-based hydrogels as novel tools in wound dressings for their application as wound repairing agents. Furthermore, gamma radiations were successfully employed for the synthesis of silver nanoparticles comprising of alginate and polyvinyl pyrrolidone (PvP)-based hydrogels. These products have scientifically shown their capability regarding the prevention of fluid accumulations in exudate wounds [177]. The amalgamation of nano-silver particles provides a promising anti-microbial property and hence made these PvP-alginate hydrogels most appropriate for wound healing and dressing. Other than alginate and their associated derivatives, numerous other naturally occurring polysaccharides like hyaluronic acid, cellulose, chitosan, and chitin have been investigated by researchers to assess their wound healing applications [178].

#### *4.12. Drug Delivery and Controlled Release*

Application of bioactive polysaccharides as a novel agent in drug delivery and controlled release has also been studied by scientists owing to their least toxicity, minimum immunogenicity, and biocompatibility. Various naturally occurring polysaccharide-based drug delivery systems are in practice due to their targeted delivery/controlled release, shielding effect against premature degradation of drugs, improvement of intracellular transportation, enhancement of bioavailability of drugs, as well as delivery of small interfering RNA, antigens, and genes [179]. Delivery systems mentioned here usually possess covalent/ionic cross-linkages, poly-electrolyte complexes, conjugates of polysaccharides and drugs, and self-assembly [179]. Release of 3-D cross-linked drugs could be triggered by varying redox potential, pH, light, ions, temperature, and application of magnetic and/or electric fields [180]. Mainly the three most abundantly used polysaccharides i.e., alginate, chitin, cellulose, and chitosan are overviewed in detail as under in this portion.

Pharmaceutic application of cellulose and their associated derivatives could be classified either as pharmaceutical excipients for protecting purposes or as bioactive molecules themselves. Application of bioactive polysaccharides as pharmaceutical excipients in orally administrated drug delivery systems have been explored to enhance the solubility and bioavailability of drugs, to increase the final product (drug) stability, and to attain release profile from final formulations [181]. These days, microcrystalline cellulose, rice, and corn starches have been broadly engaged in formulations of capsule diluents, tablet dis-integrants, and glidants. Various cellulose derivatives like HPMC (hydroxypropyl methyl cellulose), MC (methyl cellulose), HPC (hydroxypropyl cellulose), and HEC (hydroxyethyl cellulose) possessing better physiochemical properties as compared to cellulose are evidently being used in pharmaceutical industries [182]. For instance, HPMC phthalate has significant pH depending solubility, specifically, stability under acidic conditions of the stomach while soluble in mild acidic to slight alkaline solutions and, hence, are being applied for controlled release of intestinal targeted drugs. In recent times, nanocellulose-based drug delivery systems comprising of CNCs (cellulose nanocrystals), NFC (nanofibrillated cellulose), and BC (bacterial cellulose) have been investigated comprehensively [183]. For example, the binding and release of the hydrochloride salt of doxorubicin and tetracycline have been explored extensively due to ionic cross-linked systems, in which sulfate groups on cellulose nanocrystals possessing negative charge are reversibly cross-linked ionically to counterpart positively charged drugs. Likewise, nanofibrillated cellulose-based films have also been investigated for entrapment of drugs and are being used in pharmaceutical industries for the production of long-lasting drug release systems [184].

Reconnoitering the application of chitin/chitosan as bio-molecular delivery vectors have impelled the scientists for the development of therapeutic drug delivery systems like siRNA (small interfering RNA) carriers, antigens, and genes [185]. In vivo, therapeutic application of chitosan-based siRNA carries has shown great potential as a tool for gene expression associated diseases. Inhibitory influence on human colorectal cancer gene expression due to the application of chitosan-siRNA nanoparticles have been studied in an earlier study [186]. It was noticed that chitosan-siRNA nanoparticles developed by ionic gelation with Na-tri-polyphosphate demonstrated a more targeted dene inhibiting impact owing to increased binding and loading effectiveness. Long-lasting delivery of encapsulated antigens or intra-dermal vaccines administrated through chitosan microneedles transdermal delivery systems are documented to deliver more sustainable immune stimulation [187]. Though, the sensitivity of pH could also affect the stability issues of the drug delivery systems [179]. Various other bioactive polysaccharides like chondroitin, pectin, xanthan gum, dextran, chitin, gellan gum, chitosan, and dextran are also being used for controlled drug delivery [1,181].

#### **5. Conclusions**

Bioactive polysaccharides have acquired significant attention from scientists as functional biomolecules for the development of innovative and value-added products in the fields of pharmaceutics, food, cosmetics, and the biomedical industry. Their therapeutic application is mainly due to their bio-degradable, non-toxic, and bio-compatible nature. Extraction and isolation of naturally occurring bioactive polysaccharides possessing high purity with maximum extraction yield, meanwhile keeping in view that the native structure remains intact, are of great future concern and remains a field for further exploration. Momentous results to authenticate the use of these polysaccharides as a novel tool in the pharmaceutical and medicinal industry will require a multidimensional approach from scientists of various fields like healthcare, food science, organic chemistry, material science and engineering, as well as plant biology.

**Author Contributions:** S.U. and A.A.K. drafted this manuscript; Y.S. edited and reviewed the whole manuscript and provided suggestions to main authors with critical input and corrections; F.S. assisted in locating and interpreting the literature sources whenever or/and wherever was necessary; and all authors read and approved the final manuscript.

**Funding:** This work was supported by the National Natural Science Foundation of China (Grant Nos. 31670064 and 31271812), and TaiShan Industrial Experts Program.

**Conflicts of Interest:** Authors declare no conflict of interest.

#### **References**

© 2019 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 (http://creativecommons.org/licenses/by/4.0/).

## *Article* **Characterization of Fulvic Acid Beverages by Mineral Profile and Antioxidant Capacity**

#### **Monika Swat, Iga Rybicka \* and Anna Gliszczy ´nska-Swigło ´**

Institute of Quality Science, Pozna ´n University of Economics and Business, al. Niepodległo´sci 10, 61-875 Pozna ´n, Poland<EMAIL_ADDRESS>(M.S<EMAIL_ADDRESS>(A.G.-S.) ´

**\*** Correspondence<EMAIL_ADDRESS>Tel.: +48-61-8-56-93-68

Received: 1 October 2019; Accepted: 18 November 2019; Published: 22 November 2019

**Abstract:** The main purpose of the study was to investigate the quality of fulvic acid-based food products. The concentrations of Ca, K, Mg, Na, Cu, Fe, Mn, and Zn, and antioxidant capacities of fulvic acid concentrates and ready-to drink beverages available on the global market were determined. The concentrations of minerals were determined using microwave plasma-atomic emission spectrometry. Antioxidant capacity was expressed as total polyphenol (TP) and flavonoid (TF) contents, the trolox equivalent antioxidant capacity (TEAC) and ferric reducing ability of plasma (FRAP) values. The daily portion of eight out of 14 products realized 45–135% of recommended daily allowance (RDA) for Fe. One of ready-to-drink beverages was also a good source of Mg (about 40% of RDA), and another one of Mn (about 70% of RDA). The concentrations of TP and TF in ready-to-drink beverages varied from 6.5 to 187 μg/mL, whereas in concentrates, from 5886 to 19,844 μg/mL. Dietary supplements or food products with fulvic acids may be a good source of antioxidant polyphenolic compounds and some minerals.

**Keywords:** antioxidant capacity; fulvic acids; functional beverage; iron; mineral

#### **1. Introduction**

Fulvic acids are natural, water-soluble polymers, which are the ingredients of humic substances defined as "a series of high molecular weight substances, yellow to black in colour, formed as a result of secondary synthesis reactions" [1,2]. They are complex substances without standard chemical formulae, which are present in soil and plants in trace amounts [3,4]. They are formed during the decomposition of decaying plants by microorganisms and they play essential functions in plants; e.g., are responsible for the absorption of nutrients and trace substances. Naturally, fulvic acids contain minerals (more than 70), amino acids, sugars, peptides, nucleic acids, phytochemical compounds, vitamins, and fragments of plant DNA [3]. Most of them occur in ionic form. This means that fulvic acids conduct electricity excellently and improve the absorption of other compounds interacting with them. Moreover, because of ionic minerals, fulvic acids help to increase their bioavailability in plants [5]. Fulvic acids are also chemically reactive because of the presence of many carboxyl and hydroxyl groups [3]. Due to their low molecular weights, they can transport minerals to plant cells in the root, stem, and leaves [6]. They also participate in the carbon cycle, because they are constantly recycled among plants, soil, and water [7].

The results of the studies on fulvic acid properties conducted with plants and plant cells indicate the positive effect of these substances on animal organisms. Kishor et al. [8] suggested that humic substances, consisting of 60–80% fulvic acids, have anticarcinogenic properties. They may be beneficial in cancer therapy due to their heavy metal chelating properties, binding of proteins delivering anticancer drugs, and inhibition of cancer cell proliferation [9–11]. Fulvic acids are also good free radical scavengers. They may scavenge superoxide (O2 •−), hypochlorous acid (HOCl), hydrogen peroxide (H2O2), hydroxyl radicals (•OH), peroxynitrite (ONOO−), and singlet oxygen (1O2) [12,13]. Carrasco-Gallardo et al. [14] analysed the results of various clinical studies confirming the effect of fulvic acid consumption on the reduction the symptoms of Alzheimer's disease. Their studies suggested that fulvic acids can be potentially used to prevent this brain disorder, mainly due to their antioxidant properties. Many studies have provided the evidence that accumulation of oxidative stress products in brain tissue is closely associated with the development of Alzheimer's disease and antioxidant therapies are one of the promising therapeutic strategies for this disease [15]. It is thought that fulvic acids improve the absorption of iron, making it more bioavailable to bone marrow stem cells for formation of blood [16]. Van Rensburg [17] and Winkler and Gosh [18], in their reviews, listed few studies conducted with both animals and humans over several years on the safety of fulvic acids and their effects on human diseases. In these studies, fulvic acids were applied orally and topically. The studies were carried out using various doses and forms of fulvic acids, and with various durations. The results obtained suggested that fulvic acids are safe for humans; nevertheless, further studies to ensure their safety are still required. The recommended daily dose of fulvic acids for people is not established. Therefore, it is important to determine the optimal dosage of fulvic acids for different age groups to prevent them from overdosing.

Fulvic acids have been primarily used as products supporting the growth of plants and maintaining soil moisture. Currently, the food market has also become interested in them. Considering all the properties of fulvic acids, there is a potential to use them as a new, natural, and valuable food additives or supplements [19]. Due to the structure of fulvic acids and their chelating properties they can help to transport some nutrients, mainly minerals, to cells and remove deeply embedded toxins from the body [16]. Substances constituted up to 20% by fulvic acids have been used in traditional Indian medicine, "Ayurveda," for medicinal purposes for about 3000 years [20,21]. Fulvic acids intended for human consumption are currently available in concentrated form, ready-to-drink beverages and pills, but their variety on the food market is still small. These products are a potential source of minerals and antioxidant compounds. As far as is known, there are no data characterizing fulvic acid products in the aspects of either mineral profile or antioxidant capacity. Therefore, the objective of this study was to determine the content of selected macroelements and microelements and the antioxidant properties of fulvic acid beverages available on the global market.

#### **2. Materials and Methods**

#### *2.1. Materials*

The products were bought in 2015 in on-line stores. The analyses were performed for most of fulvic acid beverages available at that time on the global market. The experiments were repeated for the same products bought in 2018. The total of 14 beverages were included in the study—6 concentrates and 8 ready-to-drink beverages. According to the label description, fulvic acids in concentrated form originated from the Great Salt Lake in Utah (North America) and England. Fulvic acids in ready-to-drink beverages were obtained mainly from an aquatic source and/or soils of North America and South Africa.

#### *2.2. Sample Preparation*

For determination of minerals, the products tested were diluted with demineralized water (Hydrolab System, Wi´slina, Poland). The dilution of ready-to-drink beverages was 10-fold and that of concentrates was 100-fold.

For determination of total phenolics (TP), total flavonoids (TF), and the antioxidant capacities, the products were centrifuged at 14,000× *g* for 5 min (MiniSpin plus centrifuge, Eppendorf, Hamburg, Germany) and diluted with demineralized water if necessary.

#### *2.3. Determination of Minerals*

The concentrations of Ca, K, Mg, Na, Cu, Fe, Mn, and Zn were determined using microwave plasma-atomic emission spectrometry (Agilent MP-AES 4210) (Agilent Technologies, Melbourne, Australia) according to the method described in detail by Ozbek and Akman [22]. The analytical wavelengths and standard curves for minerals we analysed were: 616.217 nm and 0–50 μg/mL for Ca, 404.414 nm and 0–500 μg/mL for K, 383.829 nm and 0–20 μg/mL for Mg, 330.237 nm and 0–100 μg/mL for Na, 324.754 nm and 0–0.1 μg/mL for Cu, 371.993 nm and 0–10 μg/mL plus 259.940 nm and 10–100 μg/mL for Fe, 403.076 nm and 0–10 μg/mL for Mn, and 213.857 nm and 0–5 μg/mL for Zn. For each determination, at least two calibration curves were prepared, each adjusted to the expected concentration in the sample being analysed. Six determinations were performed for each sample.

#### *2.4. Determination of Total Phenolics*

The concentration of TP was determined according to Singleton and Rossi [23]. The method was adapted to 48-well microplates. In brief, 0.01 mL of each sample was mixed with 0.05 mL of the Folin–Ciocalteu reagent. After 3 min, 0.15 mL of 20% sodium carbonate and 0.79 mL of demineralized water were added and the solution was mixed. The plate was incubated for 2 h in the dark at room temperature. The absorbance was measured at 765 nm. At least six determinations were performed for each sample. The total content of phenolics was expressed in μg of gallic acid per millilitre of product.

#### *2.5. Determination of Total Flavonoids*

The concentration of TF was determined according to Karadeniz et al. [24]. The method was adapted to 48-well microplate and (±)-catechin was used as the standard. In brief, 0.10 mL of each sample was mixed with 0.50 mL of demineralized water and 0.03 mL of 5% NaNO2. After 5 min, 0.06 mL of 10% AlCl3 and 0.2 mL of 1M NaOH were added. After 5 min, 0.11 mL of demineralized water was added and the solution was mixed. The absorbance was measured at 510 nm and corrected for the absorbance of product sample and the absorbance of blank sample. At least six determinations were performed for each sample. The total content of flavonoids was expressed in μg of (±)-catechin per millilitre of the product.

#### *2.6. Determination of the Antioxidant Capacity*

The antioxidant capacity of fulvic acid products was determined using the TEAC method with ABTS•+ radical cation as described by Re et al. [25]. Moreover, the FRAP assay was carried out by the method of Benzie and Strain [26] with modifications previously described [27].

Briefly, the ABTS•+ radical cation was generated by reaction of 0.0077 g of ABTS dissolved in 1.8 mL of demineralized water with 0.2 mL of 0.0066 g/mL potassium persulphate. The reaction mixture was incubated in the dark at room temperature for 16 h [25]. The ABTS•+ radical cation working solution was obtained by dilution with methanol to an absorbance about 0.80 at 734 nm. The absorbance was measured 6 min after mixing 0.008 mL of sample with 0.792 mL of the ABTS•+ radical cation working solution. The TEAC value was calculated as the ratio of the linear regression coefficient of the calibration curve for five dilutions of the sample and the linear regression coefficient of the trolox standard curve. Three independent determinations were performed for each sample. The activity of each product was expressed as the TEAC value (in μmol of trolox/mL of product).

The FRAP assay is based on the reduction of a ferric 2,4,6-tripyridyl-s-triazine complex (Fe3+-TPTZ) to the ferrous form (Fe2+-TPTZ) in the presence of antioxidant. A volume of 0.008 mL of sample was added to 0.792 mL of the 10 mM ferric-TPTZ reagent and the increase in absorbance at 593 nm was measured after 8 min. The FRAP value was calculated as the ratio of the linear regression coefficient of the calibration curve for five dilutions of the sample and the linear regression coefficient of the FeSO4 × 7 H2O standard curve [27]. Three independent determinations were performed for each sample. Activity of each product was expressed as the FRAP value (μmol of Fe2+/mL).

#### *2.7. Statistical Analysis*

Statistical analyses were carried out using Statistica 12.0 software (2013; StatSoft, Inc., Tulsa, OK, USA). All data were submitted to one-way analysis of variance (ANOVA). The significances of differences between mean values obtained for products were determined by the least significant differences tests at α = 0.05. The comparison of data in Tables 1–3 is category-separated for clarity of data presentation.

*Foods* **2019** , *8*, 605

*Foods* **2019** , *8*, 605

*Foods* **2019** , *8*, 605

#### **3. Results and Discussion**

Each producer of a fulvic acid beverage declared that they used a standard, repeatable, and controlled process to manufacture each batch of their product(s). On the other hand, they usually stated that some differences in the concentration of fulvic acids and individual trace substances may be observed because the starting material is a completely natural material, not subjected to any laboratory treatment. Because of this, products from different years (2015 and 2018) were regarded as independent samples. As products selected were from different categories (concentrates and ready-to-drink beverages); all results were expressed both per millilitre of the product and in regard to daily intake calculated for the maximum portion suggested by producers.