**Qualitative and Nutritional Improvement of Cereal-Based Foods and Beverages**

Editors

**Antonella Pasqualone Carmine Summo**

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

*Editors* Antonella Pasqualone Department of Soil, Plant and Food Science (DISSPA), University of Bari Aldo Moro Italy

Carmine Summo Department of Soil, Plant and Food Science (DISSPA), University of Bari Aldo Moro Italy

*Editorial Office* MDPI St. Alban-Anlage 66 4052 Basel, Switzerland

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/cereal foods).

For citation purposes, cite each article independently as indicated on the article page online and as indicated below:

LastName, A.A.; LastName, B.B.; LastName, C.C. Article Title. *Journal Name* **Year**, *Volume Number*, Page Range.

**ISBN 978-3-0365-0706-4 (Hbk) ISBN 978-3-0365-0707-1 (PDF)**

Cover image courtesy of Antonella Pasqualone.

c 2021 by the authors. Articles in this book are Open Access and distributed under the Creative Commons Attribution (CC BY) license, which allows users to download, copy and build upon published articles, as long as the author and publisher are properly credited, which ensures maximum dissemination and a wider impact of our publications.

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### **Contents**

### **About the Editors**

**Antonella Pasqualone** is an Associate Professor of Food Science and Technology at the University of Bari, Italy. Scientifically responsible for many national and international projects, she published 186 articles in refereed international scientific journals, with an h-index of 33 (February 2021). Her main research interests are in the field of cereal science and technology: (i) improvement of the nutritional quality of cereal-based foods by integration with sustainable crops such as pulses; (ii) formulation and characterization of functional pasta and bakery products, also gluten-free; (iii) studies on the baking systems of flatbreads; (iv) improvement of bread shelf-life; (v) activity of polyphenol oxidase in durum wheat and correlations with pasta browning; (vi) effect of drying or baking on the volatile profile of pasta and bread; (vii) food upcycling by reusing food industry waste and by-products in cereal-based foods; (viii) rediscovery and qualitative standardization of non-alcoholic fermented cereal beverages and traditional cereal-based foods.

**Carmine Summo** is an Associate Professor of Food Science and Technology at the University of Bari, Italy. Scientific coordinator and Research Unit member for several research projects, he published 155 articles in refereed international scientific journals, with h-index = 26 (February 2021). His main research interests fall in the field of cereals and legume-based foods: (i) formulation and characterization of functional pasta and bakery products containing bioactive extracts from food industry by-products and waste; (ii) quality characterization of pasta and bakery products, also gluten-free; (iii) improvement of the nutritional quality of cereal-based foods by adding legume flours; (iv) studies on the effect of drying on the volatile compounds of pasta; (v) formulation and characterization of innovative ready-to-eat legume-based foods; (vi) chemical, nutritional and functional characterization of legume varieties to increase the knowledge on the legume properties and avoid the risk of genetic erosion; (vii) studies on the application of air classification to legume flours and on food application of the derived fractions; (viii) production of meat analogues from cereals and pulses flours.

### *Editorial* **Qualitative and Nutritional Improvement of Cereal-Based Foods and Beverages**

#### **Antonella Pasqualone \* and Carmine Summo**

Department of Soil, Plant and Food Science (DISSPA), University of Bari Aldo Moro, Via Amendola, 165/a, I-70126 Bari, Italy<EMAIL_ADDRESS>
**\*** Correspondence<EMAIL_ADDRESS>
Received: 28 January 2021; Accepted: 3 February 2021; Published: 5 February 2021

**Abstract:** The main directions of research aimed at nutritional improvement have to face either excesses or deficiencies in the diet. To this end, different strategies may be adopted, such as the reformulation of products, the introduction of functional ingredients, and the application of biotechnology to increase the bioavailability of bioactive compounds. These interventions, however, can alter the physico-chemical and sensory properties of the final products, making it necessary to achieve a balance between nutritional and quality modification. This Special Issue offers readers information on innovative ways to improve the cereal-based foods and beverages, useful for researchers and for industry operators.

**Keywords:** functional foods; upcycling; byproducts; bioactive compounds; dietary fiber; new quality; pulses; insects; bread; pasta

Increased consumer awareness of the effects of food in preventing nutrient-related diseases and maintaining physical and mental well-being, has made nutritional improvement an important goal of the food and beverage industry, including the cereal sector. To this end, different strategies may be adopted, such as the reformulation of products, the introduction of functional ingredients, and the application of biotechnologies to increase the bioavailability of bioactive compounds. These interventions, however, can alter the physico-chemical and sensory properties of the final products, making it necessary to achieve a balance between nutritional and quality modification.

The Special Issue "Qualitative and Nutritional Improvement of Cereal-Based Foods and Beverages" collects 17 original research articles and one review aimed at exploring innovative ways to improve cereal-based foods and beverages, an old—if not ancient—group of products which are still on our table every day.

In these articles, a wide range of very different food products is considered, such as bakery products (including white bread, brown bread, durum wheat bread, tortilla, pizza base, muffins and biscuits), fresh and dry pasta, extruded sticks and instant flours, fortified blended foods and Sunsik, the latter being a traditional Korean beverage [1]. Cereal-based beverages, indeed, hold a long tradition and have become known for their sensory and health-promoting attributes [2].

The main directions of research aimed at nutritional improvement have to face either excess or deficiency in the diet. In the latter case, nutrient-rich foods with long shelf-life are needed to prevent malnutrition, whereas in developed countries it is mostly required to decrease the energy value, sucrose, salt, and increase dietary fiber content of foods to prevent obesity and nutrient-related chronic diseases such as cardiovascular disease, hypertension, and diabetes mellitus. The 2030 Agenda for Sustainable Development, adopted in 2015 by the United Nations, set the goals "*to end hunger, achieve food security and improved nutrition, and promote sustainable agriculture*" (Goal 2) and "*to ensure healthy lives and promote*

*Foods* **2021**, *10*, 338

*well-being for all at all ages*" (Goal 3), recognizing non-communicable diseases (NCDs) as a major challenge for sustainable development [3]. In addition, patients with obesity and other chronic underlying conditions are at particularly high risk of developing severe COVID-19 complications [4].

The most natural way to improve the nutritional profile of cereal-based foods is to use wholemeal flour, thus retaining all fiber and micronutrients of wheat caryopsis. Wholewheat flour is a valuable raw material, irrespective of the milling system (stone milling or roller milling) [5]. Higher contents of bioactives can be reached if purple wheat, whose debranning fractions are particularly rich in anthocyanins, is used [6]. Fermentation further improves the nutritional features of wholemeal flours. Fresh pasta prepared by mixing semolina with wholewheat sourdough shows higher content of free essential amino acids and phenolic compounds, lower phytic acid content, and higher antioxidant activity, than control pasta where non-fermented wholewheat semolina is used [7].

Another strategy consists in adding locally available ingredients to reformulate existing cereal-based foods, in order to increase the nutritional value while diminishing the risk of genetic erosion of the local crops and reduce imports. In this context, the leaf powder of *Moringa oleifera*, a plant originating in India and Africa, which is rich in proteins, minerals, and phytochemicals, has been proposed as an additional ingredient to improve the nutritional profile of white and brown bread [8] or fresh pasta [9]. The fortification with moringa leaf increases protein and iron content, but makes bread darker lowering the consumer acceptability, although with a minor impact on brown bread. The addition of moringa leaf powder to fresh pasta in the range 5–15% significantly increases the content of polyphenols even at the lowest percentage. Similarly, legumes can be used which are rich in proteins and complement the amino acid profile of cereal-based foods. Flour of Apulian black chickpeas, an autochthonous black-coated chickpea cultivated in Southern Italy, rich in anthocyanins, has been added to various bakery products [10], namely bread, "focaccia"—an Italian traditional bakery product similar to pizza [11], and pizza crust by substituting flour in the 10–40% range. The rheological properties of dough worsen, resulting in harder and darker final products. However, the nutritional features improve in terms of higher contents of fiber, proteins, and bioactive compounds, as well as higher antioxidant activity [10]. In pasta, the addition of chickpea and hemp flour, previously fermented and enzymatically treated, improves the nutritional profile and protein digestibility, and reduces the sensory drawbacks and the antinutrients (tannins, phytates and raffinose) [12].

Red quinoa or Taiwan djulis (*Chenopodium formosanum* Koidz.) can be used to develop sourdough bread (20% djulis sourdough and 80% wheat flour) [13], whereas germinated wheat, in combination with an extract of *Achyranthes aspera* and *Acanthopanax*, two plants used in Asian traditional medicine, has been proposed to fortify Sunsik, a traditional ready-to-drink Korean cereal-based beverage made of roasted brown rice, barley, adlay, oat, and black beans [1]. Amaranth (*Amaranthus hypochondriacus* L.) and flaxseed (*Linum usitatissimum* L.), instead, have been added to corn-based instant-extruded products, to meet the needs of nutritionally balanced ready-to-eat foods. The addition effectively increases lysine, polyunsaturated fatty acids, minerals, and fiber of the end-products [14]. Seed flour of *Brosimum alicastrum* Sw., a Mexican tree locally named "ramón," characterized by high protein, dietary fiber, and micronutrient content, has been added to wheat tortillas improving the healthy features while keeping pliability unaltered, but with a browner color [15].

Insects are another valuable source of proteins which could be used to overcome the challenge of a more sustainable food chain while improving the nutritional profile of end-products. Meal of winged termites (*Macrotermes bellicosus*) has been added to biscuits prepared with sorghum and wheat flour [16]. Sorghum, which can be grown in tropical areas, makes this biscuit formulation viable in sub-Saharan areas, where protein-energy malnutrition is a major health concern. A significant increase of proteins, minerals, and amino acids is achieved, but biscuits become darker and less hard [16]. In the same geographic area, fortified blended foods (corn-soy blends or wheat-soy blends) are used to prepare viscous porridges in

*Foods* **2021**, *10*, 338

supplementary feeding programs [17]. These food aids often result in products too viscous for being fed to infants and young children, therefore it is needed to dilute them, lowering the nutritional value and energy density. The addition of cowpea has been proposed, also in combination with extrusion cooking, to obtain a porridge able to deliver the correct amount of nutrients at lower viscosities [17]. Extruded cooked products are rehydrated easily without cooking [18], which is important to save energy sources.

The use of waste and byproducts from the food industry, where upcycling is becoming increasingly important to improve sustainability, represents another mean for enhancing the nutritional and healthy features of cereal-based foods [19]. Coffee silverskin, a byproduct from the coffee industry rich in dietary fiber, proteins and bioactive compounds, has been used to produce extruded sticks based on cornmeal and amaranth flour [20], whereas almond skins, a byproduct of almond-based confectionery industry, rich in fiber and phenolic compounds, have been effectively considered as a functional ingredient in biscuits, which become more friable but darker [19].

Finally, to meet the needs related to western lifestyle, the production of muffins containing agave syrup instead of sucrose [21], and the reduction of the sodium content of bread by using a natural low-sodium sea salt [22], have been proposed.

To sum up, this Special Issue gives an interesting contribution to the field and offers readers information on several ways to innovate and improve the cereal-based foods and beverages, which can be useful both for researchers and for industry operators. In most cases, the reformulation with additional ingredients alters the sensory properties, therefore raising the need of communicating a "new quality" to consumers to explain that the differences with conventional counterparts are largely compensated by improved nutritional features.

**Author Contributions:** A.P. writing—original draft preparation; A.P., C.S. writing—review and editing; A.P., C.S. funding acquisition. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was funded by: (1) Agropolis Fondation, Fondazione Cariplo, and Daniel and the Nina Carasso Foundation through the "Investissements d'avenir" program with reference number ANR-10-LABX-0001-01, under the "Thought for Food" Initiative (project "LEGERETE"); (2) the Italian Ministry of Education, University and Research (MIUR), program PRIN 2017 (grant number 2017SFTX3Y) "The Neapolitan pizza: processing, distribution, innovation and environmental aspects."

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

#### **References**

#### *Foods* **2021**, *10*, 338

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© 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

#### *Article*

### **Characteristics of** *Sunsik***, a Cereal-Based Ready-to-Drink Korean Beverage, with Added Germinated Wheat and Herbal Plant Extract**

#### **Bo Ram Kim 1, Seung Soo Park 1, Geum-Joung Youn 2, Yeon Ju Kwak 2 and Mi Jeong Kim 1,3,\***

Received: 16 October 2020; Accepted: 6 November 2020; Published: 12 November 2020

**Abstract:** The purpose of this study was to develop a formulation of *Sunsik* with improved health benefits by adding germinated wheat (GW) and herbal plant extract (HPE) using a response surface methodology (RSM). The central composite experimental design (CCD) was used to evaluate the effects of *Sunsik* with added HPE (2–4%) and GW (10–20%) on total phenolic content (TPC), total flavonoid content (TFC), Trolox equivalent antioxidant capacity (TEAC), 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging capacity, gamma butyric acid (GABA) content, total color changes (-E), browning index (BI), water absorption index (WAI), and water solubility index (WSI). As a result of the CCD, the independent and dependent variables were fitted by the second-order polynomial equation, and the lack of fit for response surface models was not significant except in relation to WSI. The GABA content, TPC, and TEAC were more adequate for a linear model than for a quadratic model, and they might be affected by GW rather than HPE. Alternatively, the TFC, DPPH radical scavenging capacity, WAI, WSI, -E, and BI were fitted with quadratic models. The optimum formulation that could improve antioxidant and physicochemical properties was *Sunsik* with 3.5% and 20% added HPE and GW, respectively.

**Keywords:** cereal-based ready-to-drink beverage; convenient meal replacement (CMR); germinated wheat; response surface methodology (RSM); gamma-amino butyric acid (GABA); antioxidant properties

#### **1. Introduction**

Recently, the increase in single-person and double-income households has shifted consumers' eating behaviors toward the increased consumption of home meal replacements (HMRs) or convenient meal replacement (CMRs) [1]. As ready-to-eat foods, CMRs are a more convenient and simpler meal replacement than HMRs, and they could reduce meal preparation and eating time. The CMR market quadrupled from \$600 million in 2009 to \$2.3 billion 2019. In Korea, the proportion of single-person households is expected to reach 35% of the total population in 2030, and the CMR market is expected to continue to grow.

The types of CMR products are diversifying, such as to include liquid and powder grains, porridges, and cereal bars. Among them, cereal-based beverages are a representative CMR product consumed worldwide because they provide an efficient means to increase the intake of essential nutrients among busy modern people. A few studies investigated the physicochemical and health-conscious properties of various cereal beverages [2,3]. Bembem and Agrahar-Murugkar [2] reported that

millet-based ready-to-drink beverages improved radical scavenging activity, total phenolic content (TPC), and viscosity in the geriatric population. In another study, multigrain beverages prepared with barley, oats, buckwheat, and red rice were identified as providing additional health benefits, such as phenolic content and soluble fiber, to consumers [3].

*Sunsik* has been consumed for a long time as a cereal-based ready-to-drink beverage in Korea. It is made of partially raw or thermal-processed and dried agricultural and marine products [4]. The most common ingredients of *Sunsik* are roasted brown rice, barley, adlay, oat, and black beans [5]. With the recent increase in the demand for healthy foods, much research has reported that additional ingredients, such as various dried vegetables, nuts, and fruits, could be added to *Sunsik* to offer more health-conscious nutrients [6–8]. For example, Park [8] reported that *Sunsik* with added mealworm was higher in antioxidant capacities and in consumer preference than a control *Sunsik*. Regarding the quality of ready-to-drink of *Sunsik*, it should disperse and dissolve well in water or milk within a few minutes. Koh, Jang, and Surh [6] reported that fermented *Sunsik* had a higher soluble solid content, oxidative stability, and amino acids than unfermented *Sunsik*, resulting in an improved solubility and nutrient content. Although several studies reported enhancements in the quality and nutrient content of *Sunsik*, there is limited information on the health benefits of *Sunsik* with added germinated wheat (GW) and herbal plant extract (HPE).

Germination has been identified as an effective processing method to improve the nutritional quality and health-related compounds of cereal [9]. In numerous studies, gamma amino butyric acids (GABA) and phenolic acid compositions were increased as the germination time of wheat increased, suggesting the possibility of GW as a health-conscious ingredient [10–12]. In addition, Dhillon et al. [13] found that the antioxidant activity of and consumer preference for breads were improved when GW flour at 30 ◦C for 72 h was partially used to make bread. The changes in the physiological and biochemical properties of GW might be due to the activation of endogenous enzymes that break down starch and protein into small molecules [14,15]. The activation of endogenous enzymes may also play a role in increasing the solubility of *Sunsik* with added GW when it mixes with water or milk. In addition, plant herbal medicines, such as *Achyranthes aspera*, safflower seed, and Acanthopanax, have been used for the prevention of various diseases in traditional treatments in Asian countries [16,17]. It is known that safflower seeds are rich in lignin, flavonoid, and serotonin and have excellent effects on bone diseases, such as osteoporosis [18]. As previously published in many studies, the extracts of *A. aspera* and *Acanthopanax* showed a reduced inflammatory effect and antioxidant capacities [19–22]. The above-mentioned herbal plant medicines are used not only for therapeutic purposes, but also by adding them to various foods in the form of extracts to increase the health-related functions in the food matrix, such as noodles, drinks, and cookies [23–26]. The HPEs, including *A. aspera*, safflower seed, and *Acanthopanax*, used in this study confirmed previously the pharmacological effects on osteogenic differentiation in human mesenchymal stem cells [27]. The mixture extracts of herbal plants were freeze-dried and then were used in various food products of Gagopa Healing Food Co., Ltd. (Changwon, Korea).

Currently, *Sunsik* with added GW flour and HPE is not available in the marketplace yet. Thus, if GW and HPE are added to commercial *Sunsik*, which is conveniently used as ready to drink beverage, the new *Sunsik* product might be more beneficial to health. The purpose of this study was to determine the optimum formula amounts of GW flour and HPE powder for new *Sunsik* products as cereal-based ready-to-drink beverages. To determine the optimum formulation of *Sunsik*, the response surface methodology (RSM) was adopted using a central composite experimental design (CCD). The antioxidant capacities, GABA, water absorption index (WAI), water solubility index (WSI), total color changes (ΔE), and browning index (BI) were analyzed to optimize the health-conscious nutrients and quality of *Sunsik*; then, the newly optimized *Sunsik* was compared with control *Sunsik* in terms of various health-conscious and physicochemical properties.

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

#### *2.1. Materials*

The *Sunsik* and HPE were provided from Gagopa Healing Food Co., Ltd. (Changwon, Korea). The main ingredients of *Sunsik* consisted of 30% barley, 30% brown rice, 20% adlay, 10% black bean, and 10% oat. In general, each cereal was steamed and then dry-roasted. The four roasted cereals were pulverized in a batch for a production of the Sunsik. The Sunsik used in this study is being sold on the market. Gagopa Healing Food Co., Ltd. (Changwon, Korea) found effects of HPE on osteogenic differentiation through preliminary studies, and the results already published [27]. The HPE used in this study is composed of safflower seed (85%), *A. aspera* (5%), manyprickle acanthopanax (5%), and *Kalopanax septemlobus* (5%) [27]. In addition, the GW used in this study was prepared according to preliminary experiments. Anzunbaengi wheat, which was cultivated in Jinju, Korea, was germinated at 17.6 ◦C for 46.18 h to enhance GABA. After germination, the GW was freeze-dried and then grounded to powder. To develop a cereal-based ready-to-eat beverage to enhance health-related properties, *Sunsik* was formulated with HPE and GW to maximize GABA and antioxidant capacities. The ranges of HPE and GW used in this study were 2–4% and 10–20%, respectively, and the ranges were determined based on samples of five points or more as a result of consumer acceptability (nine-point hedonic scale) of *Sunsik* with added HPE or GW, respectively.

#### *2.2. Experimental Design and Optimization of the Formulation*

The amounts of HPE and GW were optimized using a CCD of an RSM [28]. The independent values were studied at five different levels (− α, −1, 0, + 1, and + α), and the actual levels are presented in Table 1.

**Table 1.** The coded levels and actual values of 13 experiments formulated with a central composite design (CCD).

Table 1 and they were evaluated to maximize the GABA, total flavonoid content (TFC), TPC, 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging capacity, Trolox equivalent antioxidant capacity (TEAC), and WSI and to minimize the WAI, ΔE, and BI. The effects of the two independent variables on the responses (Y) were modeled using the response surface regression, and they were predicted by the following Equation (1) [28]:

$$\mathbf{Y}\_{\mathbf{k}} = \beta\_0 + \beta\_1 \mathbf{X}\_1 + \beta\_2 \mathbf{X}\_2 + \beta\_{12} \mathbf{X}\_1 \mathbf{X}\_2 + \beta\_{11} \mathbf{X}\_1^2 + \beta\_{22} \mathbf{X}\_2^2 \tag{1}$$

where β0 is a constant, β1 and β2 are the linear coefficients, β12 is the interaction coefficient, and β11 and β22 are the quadratic coefficients. X1 and X2 are the levels of HPE and GW, respectively. Yk is the response variable, and each response variable is as follows; Y1 = GABA (μg/g), Y2 = TFC (μg CE/g), Y3 = TPC (μg GE/100g), Y4 = DPPH (μM TE/100g), Y5 = TEAC (mM TE/100g), Y6 = WAI, Y7 = WSI, and Y8 = ΔE, Y9 = BI. To validate the linear or quadratic model, each experimental data of independent variables was compared with the predicted values using the model developed in this study.

#### *2.3. Extraction Procedure of Sunsik Samples*

In total, 5 g of each *Sunsik* sample was extracted with 80% ethanol at 65 ◦C for 2 h, and the supernatants obtained by centrifugation (5000 rpm for 30 min) were evaporated to dryness at 45 ◦C using a nitrogen evaporator (Eyela MG-2200, Tokyo Rikakikai Co. Ltd., Tokyo, Japan). The dried extract was then re-dissolved with 80% ethanol into a final volume of 5 mL. The extract was used to determine the GABA, TEAC, DPPH, TFC, and TPC.

#### *2.4. Gamma-Amino Butyric Acid (GABA)*

The GABA contents of the *Sunsik* samples were determined according to the method described in Sharma et al. [29]. In brief, 0.1 mL of each extract was mixed with 0.2 mL of 0.2 M borate buffer and 1 mL of 6% phenol reagent. Then, 0.4 mL of 7.5% sodium hypochlorite was added, and the mixture was boiled for 10 min in a water bath. The samples were immediately cooled for 5 min, and the absorbance was measured using a spectrophotometer (EMC-11D-V Spectrophotometer, EMCLAB Instruments, Duisburg, Germany) at 630 nm. The GABA was used as a standard curve and prepared with a range of concentrations from 0 to 50 mg. Results were expressed as mg/g.

#### *2.5. Total Flavonoid Content (TFC)*

TFC was determined using the methods previously described by Dahl [30]. The extract of samples (250 μL) was added to 1.25 mL distilled water, and 70 μL of 5% sodium nitrite was added to the mixture. After 6 min, 150 μL of 10% aluminum chloride was added to the mixture. After 5 min, 0.5 mL of 1 N sodium hydroxide was added to the mixture. The absorbance was measured immediately at 510 nm. Distilled water was used as a blank. Catechin was used as a standard curve and prepared with a range of concentrations from 0 to 2.5 mg. The results were reported as catechin equivalents (CE) μg/g.

#### *2.6. Total Phenolic Content (TPC)*

TPC was determined by the method described by de la Rosa et al. [31] with modifications. TPC was measured using the Folin-Ciocalteu method. In total, 100 μL of each extract was added to 2.5 mL of 10% Folin-Ciocalteu reagent, and the mixture was allowed to stand for 2 min. Then, 2 mL of 6% sodium carbonate was added to the mixture, and it was incubated at 50 ◦C for 15 min in a water bath. The absorbance was measured at 760 nm, and distilled water was used as a blank. Gallic acid was used as a standard curve and prepared with a range of concentrations from 0 to 50 mg. Results were expressed as gallic acid equivalents (GAE) mg/g.

#### *2.7. DPPH Radical Scavenging Capacity*

The determination of the effect scavenging of the DPPH radical was based on a procedure previously described by Wong et al. [32]. A 0.1 mM DPPH solution diluted with 100% methanol was prepared. In addition, 0.1 mL of the sample and 1.9 mL of 0.1 mM DPPH were mixed well. The DPPH solution was allowed to stand for 30 min at room temperature in the dark. Then, the absorbance was measured at 515 nm, and 100% methanol was used as a blank. Furthermore, 10 mM Trolox was used as a standard curve and prepared with a range of concentrations from 0 to 500 μM. Results were expressed as μmol of Trolox equivalents (TE) μmol/100 g.

#### *2.8. Trolox Equivalent Antioxidant Capacity (TEAC)*

TEAC was performed as described by Simsek and El [33], with modifications. Briefly, an ABTS+ stock solution was prepared with 7.4 mM ABTS and 2.6 mM potassium persulfate and mixed. After, the mixture was allowed to stand for 16 h at room temperature in the dark. The ABTS+ stock solution was diluted with 100% methanol to an absorbance wavelength of 0.7 at 734 nm. Then, 2960 μL of the ABTS+ stock solution was added to 20 μL of the sample, and absorbance was measured after 7 min. Trolox was used as a standard curve and prepared with a range of concentrations from 0 to 1000 μg. Results were expressed as mmol of TE mmol/100 g.

#### *2.9. Water Absorption Index (WAI) and Water Solubility Index (WSI)*

The WAI and WSI of the optimized *Sunsik* and control samples were determined using methods previously described by Du et al. [34] with slight modifications. In total, 2.5 g of the sample was added to 30 mL of distilled water and mixed in a shaking water bath at 30 ◦C for 30 min. Then, the mixture was centrifuged at 3000 rpm for 15 min. The supernatant and remaining sediment from the mixture were weighted. The supernatant was decanted into an aluminum dish and dried at 105°C overnight using a dry oven. The WAI and WSI were calculated as in the following equations, respectively.

$$\text{WAI} = \frac{\text{weightofthesediment (g)}}{\text{weightofthesample (g)}} \tag{2}$$

$$\text{WSI}(\%) = \frac{\text{weight of dry solids from the supernovaant} \left(\text{g}\right)}{\text{weight of the sample (g)}} \times 100\tag{3}$$

#### *2.10. Color Properties*

The color values of the optimized Sunsik and control samples were determined with a CIE Lab system using a color meter (CR-400, Konica minolta sensing Inc., Osaka, Japan). It was calibrated with a white ceramic plate before measuring the sample. The total color changes (ΔE) and browning index (BI) were calculated as follows [35,36]:

$$
\Delta E = \sqrt{(L\_0^\* - L^\*)^2 + (a\_0^\* - a^\*)^2 + (b\_0^\* - b^\*)^2} \tag{4}
$$

$$BI = \left[100(X - 0.31)\right] / 0.172 \tag{5}$$

$$X = (a^\* + 1.75L^\*) / (5.645L^\* + a^\* - 3.012b^\*)\tag{6}$$

where *L*∗ 0, *a*∗ 0, and *b*∗ 0 are color parameters for the control and *L*∗ , *a*∗ , and *b*∗ are color parameters for each Sunsik sample.

#### *2.11. Apparent viscosity of Sunsik Samples*

The apparent viscosity of the optimized *Sunsik* and control samples was measured using a digital rotary viscometer (WVS-0.1M, DAIHAN Scientific, Gang-Won-Do, Korea). First, 45 g of the sample was placed in a 500-mL beaker, and 300 mL of water or milk was poured in, followed by thorough mixing with a magnetic stirrer (MS-20D, DAIHAN Scientific, Gang-Won-Do, Korea). Finally, the thoroughly mixed sample was poured into a 250-mL beaker (SDS 2400, DONG SUNG science, Gang-Won-Do, Korea) and the viscosity of the sample was measured. When measuring the viscosity, the standard was measured when the torque value was close to 50%.

#### *2.12. Cell Proliferative E*ff*ects of Sunsik Samples on Caco-2 and HepG2 Cells*

In total, 15 g of the *Sunsik* samples was extracted with 80% ethanol, evaporated to dryness at 45 ◦C, and re-dissolved in dimethyl sulfoxide (DMSO) according to a previously described method [37]. The Caco-2 (ATCC®HTB-37TM, Manassas, USA) cell was cultured in MEM (Hyclone Laboratories Inc.,

South Logan, UT, USA) with 10% or 20% fetal bovine serum (FBS, Welgene, Daegu, Korea) at 37 ◦C in a humidified incubator with 5% CO2. The cell proliferation of *Sunsik* extracts was determined by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay. The cells (1 × 104/well) were seeded in 96-well plates and then allowed to attach overnight. After overnight, the media included with *Sunsik* extracts were exchanged and incubated for 72 h. After 72 h of incubation, cell proliferation was determined using the MTT Cell Proliferation Assay kit (Roche Ltd., Mannheim, Germany) at 570–655 nm with a SpectraMax®i3 plate reader (Molecular Devices, Sunnyvale, CA, USA).

#### *2.13. Data Analysis*

The Design Expert software (version 11, State-Ease Inc., Minneapolis, USA) was used to analyze the experimental data for best fit model equations and to obtain response plots for each response variable. The combination of independent variables generating the highest overall desirability was selected as the optimum formulation. To validate the optimization process, the Sunsik was prepared using the optimum levels of independent variables and analyzed for the selected responses. The absolute residual error (%) was calculated using the experimental and predicted data through the following Equation (7):

$$Absolute\ residual\ error(\%) = \frac{Actual\ value - Predicted\ value}{Actual\ value} \times 100\tag{7}$$

All experiments were carried out in triplicate, and ANOVA was performed to determine differences among the samples using the XLSTAT software (Addinsoft, Paris, France). When a difference among the samples was identified, the Student Newan–Keul's (SNK) multiple comparison was performed to separate the means.

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

#### *3.1. Fitting the Model and Statistical Analysis*

The RSM is often used to determine the formulation ratio of a new product in the food industry. In this study, a CCD was applied to determine the optimum formulation of HPE and GW to prepare healthy Sunsik, a cereal-based ready-to-drink Korean beverage. The independent and dependent variables were fitted by linear or quadratic equations, and Table 2 shows the statistical results of the regression coefficients, R2, adjusted R2, lack of fit, and p values of the fitted models on analyzed responses by CCD. As shown in Table 2, the lack of fit for response surface models was not significant without the WSI, implying that the response surface models were adequately explained for predicting the relevant responses [28].

**Table 2.** The regression coefficients, R square, adjusted R square, lack of fit, and *p* values of the fitted models on dependent variables.

\*, \*\*, \*\*\* significantly differ at *p* > 0.05, *p* < 0.01, and *p* < 0.001, respectively. β1: herbal plant extract; β2: germinated wheat.

Among the responses, GABA, TPC, and TEAC were more adequate for a linear model than for a quadratic model. Because the β2 values of GABA (*p* < 0.01), TPC (*p* < 0.05), and TEAC (*p* < 0.01) differed significantly, the GABA, TPC, and TEAC contents of newly developed Sunsik might be affected by GW rather than HPE. The final equations of GABA, TPC, and TEAC as follows:

GABA = 2.09 + 0.017 × HPE + 0.1031 × GW (8)

$$\text{TPC} = 70.57 - 0.3237 \times \text{HPE} + 2.21 \times \text{GW} \tag{9}$$

$$\text{TEAC} = 120.16 + 1.34 \times \text{HPE} + 3.39 \times \text{GW} \tag{10}$$

As described in Table 2, the TFC, DPPH, WAI, WSI, ΔE, and BI were fitted with quadratic models. The final equations of TFC, DPPH, WAI, WSI, ΔE, and BI were coded as follows:

$$\text{TFC} = 30.99 + 1.11 \times \text{HPE} + 3.03 \times \text{GW} + 1.18 \times \text{HPE} \times \text{GW} - 1.45 \times \text{HPE}^2 - 1.47 \times \text{GW}^2 \tag{11}$$

$$\text{DPPH} = 106.59 + 3.71 \times \text{HPE} + 3.32 \times \text{GW} + 2.39 \times \text{HPE} \times \text{GW} - 3.49 \times \text{HPE}^2 - 2.80 \times \text{GW}^2 \tag{12}$$

WAI = 1.85+0.0068× HPE−0.0196×GW−0.0394× HPE×GW+0.014× HPE2 +0.0263×GW2 (13)

$$\text{WSI} = 48.44 - 0.3332 \times \text{HPE} + 4.52 \times \text{GW} + 0.0882 \times \text{HPE} \times \text{GW} - 4.61 \times \text{HPE}^2 - 2.03 \times \text{GW}^2 \tag{14}$$

$$
\Delta E = 0.2224 - 0.2736 \times \text{HPE} + 0.1071 \times \text{GW} - 0.0362 \times \text{HPE} \times \text{GW} + 0.2708 \times \text{HPE}^2 + 0.1384 \times \text{GW}^2 \tag{15}
$$

$$\text{BI} = 20.02 + 0.259 \times \text{HPE} + 0.015 \times \text{GW} - 0.1831 \times \text{HPE} \times \text{GW} - 0.0531 \times \text{HPE}^2 + 0.0061 \times \text{GW}^2 \tag{16}$$

The higher values of R2 and adjusted R2 mean desirability of the model to explain the relationships between variables [28]. In this study, the responses with R2 values of 0.8 or higher were TFC, DPPH, WAI, and ΔE, indicating that the fitted equations adequately describe the effects of adding GW and HPE to Sunsik on each dependent variable.

#### *3.2. E*ff*ects of Independent Values on Health-Conscious Properties*

The GABA, TFC, and TPC contents and antioxidant capacities (DPPH radical scavenging capacity and TEAC) of differently formulated Sunsik samples by CCD are shown in Table 3. Significant differences among the 13 samples were found in the GABA (*p* < 0.01), TFC (*p* < 0.001), TPC (*p* < 0.001), DPPH (*p* < 0.05), and TEAC (*p* < 0.05) contents. The GABA content, TFC, and TPC are some of the major compounds that contribute to the antioxidant capacities, such as DPPH and TEAC [11,30,38]. The GABA content and TPC were in the ranges of 1.81–2.25 μg/g and 67–76 μg GE/100g, respectively. As shown in Table 2, the GABA content and TPC were significant in the β2 value (*p* < 0.01 for GABA and *p* < 0.05 for TPC) but not significant in the β1 value, indicating that the GABA content and TPC of Sunsik with added HPE and GW were influenced by increased GW. These results were also confirmed in the three-dimensional response surface plots of Figure 1a,c.

**Experiment No. GABA \*\* (Y1,** μ**g**/**g) TFC \*\*\* (Y2,** μ**g CE**/**g) TPC \*\*\* (Y3,** μ**g GE**/**100 g) DPPH \* (Y4,** μ**M TE**/**100 g) TEAC \* (Y5, mM TE**/**100 g) HPE (g) GW (g)** *Sunsik* **(g)** 1 1 5 44 2.01 ± 0.09 ab 26 ± 2.30 bc 71 ± 2.23 cd 96 ± 3.7 b 113 ± 3.93 b 2 2 5 43 2.00 ± 0.08 ab 24 ± 1.21 c 67 ± 0.44 f 102 ± 2.9 ab 114 ± 3.70 b 3 1 10 39 2.19 ± 0.13 a 29 ± 3.68 abc 76 ± 0.88 a 95 ± 3.1 ab 122 ± 5.48 ab 4 2 10 38 2.14 ± 0.16 a 32 ± 3.34 ab 74 ± 0.97 b 110 ± 9.2 ab 122 ± 3.30 ab 5 0.79 7.5 41.71 1.98 ± 0.06 ab 26 ± 0.92 abc 69 ± 0.70 de 96 ± 6.9 b 116 ± 4.27 b 6 2.21 7.5 40.29 2.12 ± 0.04 a 31 ± 2.80 abc 72 ± 0.09 c 103 ± 9.4 b 123 ± 2.86 ab 7 1.5 3.96 44.54 1.81 ± 0.15 b 22 ± 2.00 c 68 ± 0.40 ef 94 ± 6.9 ab 119 ± 3.60 ab

**Table 3.** The experimental values of the health-conscious variables for each independent variable.

**Table 3.** *Cont*.

All values are means of three replications ± standard deviation. Values with the same letter(s) within a column are not significantly different. \*, \*\*, \*\*\* significantly differ at *p* > 0.05, *p* < 0.01, and *p* < 0.001, respectively.

**Figure 1.** Three-dimensional response surface plots of the GABA content (**a**), TFC (**b**), and TPC (**c**). (GW: germinated wheat; HPE: herbal plant extract; GABA: gamma aminobutyric acid; TFC: total flavonoid content; TPC: total phenolic acid).

Conversely, the addition of HPE and GW had significant quadratic effects (*p* < 0.05 for β11 and *p* < 0.05 for β22) on TFC (Table 2). Figure 1b shows the three-dimensional response surface plots of TFC, implying the TFC of *Sunsik* is increased by both HPE and GW.

The antioxidant properties of 13 Sunsik samples corresponding to the experiments generated by the CCD were determined by DPPH and TEAC (Table 3). The DPPH and TEAC values of the samples differed significantly (both *p* < 0.05) and were in the ranges of 96–110 μM TE/100g and 113–127 mM TE/100 g, respectively. As presented in Table 2, the DPPH value was fitted with a quadratic model while TEAC value was fitted with a linear model. The comprehensive effects of the dependent variables (HPE and GW) on the antioxidant properties of Sunsik are represented by the response surface plots in Figure 2.

**Figure 2.** Three-dimensional response surface plots of DPPH (**a**) and TEAC (**b**).

The Sunsik samples with higher antioxidant activities contained relatively high GABA content, TPC, and TFC. These results are in agreement with previous studies [11], which reported a higher antioxidant capacity of the samples containing higher GABA content, TPC, and TFC. The increments of TPC and GABA content in Sunsik samples could be explained by the addition of GW. Chen et al. [39] reported that phenolic contents in GW increased by lignin synthesis during germination. In addition, another study explained that the GABA content in GW increased via the decarboxylation of L-glutamate [11]. Safflower seed, a major material of HPE, has protective effects against osteoporosis and a beneficial effect on atherogenic risk through various phenolic compounds, such as lignin and flavonoids [25]. Recently, the antioxidant, anti-cancer, anti-inflammatory effects of safflower seeds have been identified by a few studies [25,40,41].

#### *3.3. E*ff*ects of Independent Values on Physicochemical Properties*

The WAI and WSI are important parameters in powdered cereal-based beverages, such as Sunsik, which is eaten by dissolving in milk or water. The WAI and WSI values of the Sunsik samples tested in this study are presented in Table 4. The WAI values of the Sunsik samples were in the range of 1.82–1.95 and did not differ significantly (Table 4). Although there was no statistically significant difference in the WAI values of Sunsik samples, they tended to increase as the amount of HPE increased (Figure 3a). The WAI value of reconstituted powder, such as Sunsik examined in this study, might play a role in preventing its dissolution in milk or water [42]. As shown in the WAI results of Table 2, the linear coefficients of HPE (β1) and GW (β2) were 0.0018 and −0.0195, respectively, implying that GW in newly formulated Sunsik had a negative effect. The WSI is the amount of soluble components released from the Sunsik samples, and the values ranged from 32% to 59% (Table 4). The WSI values of Sunsik with 1.5 g of added HPE and 11.04 g of added GW were the highest among the samples, suggesting the contribution of GW to the solubility of the newly formulated Sunsik samples (Figure 3b).

**Table 4.** The experimental values of the physicochemical variables for each independent variable.

All values are means of three replications ± standard deviation. Values with the same letter(s) within a column are not significantly different. \*\*, \*\*\* significantly differ at *p* < 0.01 and *p* < 0.001, respectively.

Significant differences were observed in the ΔE (*p* < 0.001) and BI (*p* < 0.01) values among the newly formulated Sunsik samples (Table 4), which were in the ranges of 0.22–1.13 and 19.2–20.3, respectively. In the results of the regression coefficients, the HPE addition negatively affected and the GW addition positively affected the ΔE of the newly formulated Sunsik. The three-dimensional response surface plots also showed a similar trend (Figure 3c), indicating that the color of the newly formulated Sunsik was mostly affected by a higher GW amount than HPE amount. Such a result was expected, as more GW (10–20%) was added to Sunsik than HPE (2–4%). The color affects consumer

perceptions of various foods or beverages, and color changes or a brown color during processing or cooking might negatively affect consumer preferences [43]. As shown in Figure 3d, the brown color changes of Sunsik were the result of adding HPE. In a preliminary experiment to determine the range of the HPE amount, consumers tended not to prefer Sunsik with more than 4% HPE added due to its darkened color.

**Figure 3.** Three-dimensional response surface plots of the WAI (**a**), WSI (**b**), ΔE (**c**), and BI (**d**).

#### *3.4. Optimization and Validation*

Cereal-based products like Sunsik are often developed with the addition of two or more ingredients to provide additional health benefits to consumers. In this study, both GW and HPE had a significant effect on the health-related properties and physicochemical characteristics of Sunsik. The additions of GW and HPE in newly formulated Sunsik were response specific. Thus, optimization is needed to attain a formulation with the desired characteristics concerning all the responses.

*Sunsik*, a cereal-based ready-to-drink beverage, was optimized considering maximized properties, such as GABA, TFC, TPC, DPPH, TEAC, and WSI. By contrast, WAI, ΔE, and BI were minimized in Sunsik products. The optimized formula of Sunsik developed in this study was 10 g of GW, 1.79 g of HPE, and 38.21 g of Sunsik corresponding to the highest desirability of 0.719. In addition, the predicted and actual values for optimized formulations of Sunsik are presented in Table 5. Both the predicted and actual values were compared and were verified using absolute residual error values (Table 5). The errors for the responses were found to be less than 5% without ΔE. This indicated the precision of the developed and optimized regression models for the newly formulated Sunsik products.

**Table 5.** Predicted and actual values of the optimized *Sunsik* formulation.

#### *3.5. Health-Conscious and Physicochemical Properties of Optimized Sunsik*

Because the purpose of this study was to develop a newly formulated Sunsik containing GW and HPE to provide health benefits over the commercially available Sunsik, various properties of commercial and optimized Sunsik were compared. The health-conscious and physicochemical properties of both Sunsik samples are presented in Table 6. The GABA content, TPC, and TFC might be major constituents contributing to the antioxidant capacities and antiproliferative cancer cells [38]. Significant differences between the commercial and optimized Sunsik samples with respect to the GABA content (*p* < 0.001), TFC (*p* < 0.001), and TPC (*p* < 0.001) were observed (Table 6). The optimized Sunsik contained more GABA (2.23 μg/g) content, TFC (33.75 μg CE/ 100g), and TPC (73.75 μg GE/100g) than commercial Sunsik (GABA: 1.7 μg/g; TFC 19.8 μg CE/100 g; TPC: 54.4 μg GE/100g), confirming health benefits of optimized Sunsik compared to commercial Sunsik.

In addition, the DPPH (*p* < 0.001) and TEAC (*p* < 0.001) of optimized Sunsik, to which 10 g of GW and 1.79 g of HPE were added, increased significantly compared to commercial Sunsik. Numerous studies have been developed new product with more antioxidant or antiproliferative activities to contribute health benefits of consumed products [7,8,38]. According to Kim and Kim [38], cereal products containing higher phenolic or flavonoid contents had higher antioxidant capacities. In this study, optimized Sunsik contained higher TPC, TFC, DPPH, and TEAC values than the commercial Sunsik. Similar trends were observed in terms of the proliferative activities of cancer cells. The relative proliferative effects on Caco-2 and HepG2 cells after treatment with an extract of the samples are shown as the median effective dose (EC50) in Table 6. The EC50 values of optimized Sunsik for Caco-2 and HepG2 cells were 45.7 and 35.2 mg/mL, respectively. Commercial Sunsik was relatively high in EC50 values of Caco-2 (97.9 mg/mL) and HepG2 (76.2 mg/mL) cells compared to those of optimized Sunsik (Caco-2: 45.7 mg/mL; HepG2: 35.2 mg/mL), indicating relatively low antiproliferative activities. Many studies have reported that foods or beverages with antioxidant activities have cancer-protective effects [37], suggesting that cereal-based beverages could inhibit cancer cell growth. In this study, optimized Sunsik added with GW and HPE showed higher antioxidant capacity and antiproliferative activity than commercial Sunsik.

The WAI, WSI and viscosity of optimized Sunsik with added GW and HPE were compared to commercial Sunsik, and the results are shown in Table 6. The WAI and viscosity of cereal-based beverages are important quality factors [3,4]. According to the finding of Fernandes, Sonawane, and Arya [3], the high absorbing properties in cereal-based beverages resulted in increased viscosity, and high viscosity negatively affected mouthfeel and overall acceptability in sensory tests. According to the results of the current study, the WAI and viscosity of optimized Sunsik with added GW and HPE were less than that of the commercial Sunsik sample. The low WAI and viscosity might contribute to the solubility of Sunsik, which is eaten by dissolving in milk or water, showing higher WSI values in optimized Sunsik than in commercial Sunsik.

**Table 6.** Health-conscious and physicochemical properties of the optimized Sunsik formulation.

All values are means of three replications ± standard deviation. Values with same letter(s) within a row are not significantly different. \*\*\* significantly differ at *p* < 0.001.

#### **4. Conclusions**

This study showed that the CCD and RSM could be used to optimize the formulation of *Sunsik*, a cereal-based ready-to-eat beverage. RSM predicted that a *Sunsik* formula of 10 g GW, 1.79 g HPE, and 38.21 g *Sunsik* would provide a better quality with more health-conscious and physicochemical characteristics. The optimized *Sunsik* is characterized by higher GABA, TPC, TFC, DPPH, TEAC, and WAI values than commercial *Sunsik*. The EC50 of cancer cells, WAI, and viscosity were low in optimized *Sunsik* compared to commercial *Sunsik*. Overall, *Sunsik* with 10 g of added GW and 1.79 g of added HPE might increase various health-related components and biological activities while maintaining the quality of the cereal-based beverage.

**Author Contributions:** Conceptualization, M.J.K.; methodology, B.R.K., S.S.P., Y.J.K., G.-J.Y.; investigation, B.R.K., S.S.P.; data curation, B.R.K., S.S.P.; writing—original draft preparation, B.R.K.; writing—review and editing, M.J.K.; supervision, M.J.K. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was supported by Changwon National University in 2019~2020.

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

#### **References**

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### **Current Functionality and Potential Improvements of Non-Alcoholic Fermented Cereal Beverages**

**Maria Valentina Ignat 1, Liana Claudia Salant,ă 2,\*, Oana Lelia Pop 2, Carmen Rodica Pop 2, Maria Tofană 2, Elena Mudura 1, Teodora Emilia Coldea 1, Andrei Bors, a 1 and Antonella Pasqualone 3**

Received: 30 June 2020; Accepted: 30 July 2020; Published: 1 August 2020

**Abstract:** Fermentation continues to be the most common biotechnological tool to be used in cereal-based beverages, as it is relatively simple and economical. Fermented beverages hold a long tradition and have become known for their sensory and health-promoting attributes. Considering the attractive sensory traits and due to increased consumer awareness of the importance of healthy nutrition, the market for functional, natural, and non-alcoholic beverages is steadily increasing all over the world. This paper outlines the current achievements and technological development employed to enhance the qualitative and nutritional status of non-alcoholic fermented cereal beverages (NFCBs). Following an in-depth review of various scientific publications, current production methods are discussed as having the potential to enhance the functional properties of NFCBs and their safety, as a promising approach to help consumers in their efforts to improve their nutrition and health status. Moreover, key aspects concerning production techniques, fermentation methods, and the nutritional value of NFCBs are highlighted, together with their potential health benefits and current consumption trends. Further research efforts are required in the segment of traditional fermented cereal beverages to identify new potentially probiotic microorganisms and starter cultures, novel ingredients as fermentation substrates, and to finally elucidate the contributions of microorganisms and enzymes in the fermentation process.

**Keywords:** cereal beverage; fermentation; functional; non-alcoholic; health benefits

#### **1. Introduction**

Consumers' lifestyles have changed in recent years and will continue to change by being influenced by globalization, economic growth, rapid advances in food science and technology, and/or lifestyle choices and religious restrictions [1–3]. With regard to consumption behaviour, sensory acceptance is still the main choice criteria for consumers [4–6] and it is strongly dependent on cultural backgrounds, as well as previous sensory exposure to a specific food product [7,8].

Beverages are an optimum vehicle to transport nutrients and bioactive compounds into the body as well as to facilitate their bioavailability. Bioactive compounds, such as phytochemicals (e.g., phytoestrogens, phenolic compounds, flavonoids, carotenoids, etc.), dietary fibre, vitamins, fatty acids, probiotics, and minerals, can be incorporated into beverages. The presence of these compounds offers the prospect of using food as a valuable element in disease prevention strategies, particularly in the early stages of the diseases [9–11]. Worldwide, statistics clearly show the growing trend of functional beverage consumption [12], due to their nutrient contents, convenient packaging, design, ease of transportation and storage, and for their shelf-stable nature [13].

Functional beverages could be classified as dairy based, fruit and vegetable based, legume based, cereal based, coffee, or tea. The functionality traits of these beverages address different needs and lifestyles: to boost energy, to fight the ageing process, fatigue, and stress, or to target diseases [14].

Fermentation is widely used to improve the nutritional value, the digestibility level, shelf life, functional properties, texture, taste, and flavour of the beverages [15–18]. A popular class of fermented beverages are those made from cereals: barley (*Hordeum vulgare* L.), maize (*Zea Mays* L.), millet (*Panicum miliaceum* L.), oats (*Avena sativa* L.), rice (*Oryza glaberrima*/*Oryza sativa*), rye (*Secale cereale*), sorghum (*Sorghum bicolor*), and wheat (*Triticum aestivum* L.) [19]. These cereals are a good fermentation substrate and also pose a potentially functional trait, as they contain nutrients that can be easily assimilated by probiotics [20]. The processing steps could include soaking, sprouting, malting, cooking, grinding, and filtering. The fermentation of cereals is influenced by several factors (e.g., length of fermentation, temperature and pH, moisture content of grain, growth factor requirements, cereal nutrients, etc.) which require control by using technological methods to standardize quality.

Fermented beverages are affordable, and their production involves traditional methods to maintain hygiene conditions, product quality, and security [19,21]. Lactic acid bacteria (LAB) dominate the fermentation process and lead to a low pH, which is incompatible with the development of pathogenic bacteria, thereby increasing the shelf life and product safety [22]. Traditional methods exploit mixed cultures of various potentially beneficial microorganisms, referred to as probiotics [23]. Fermented beverages are considered essential because they serve as vehicles for beneficial microorganisms that play an important role in human health, and also for their nutritional, nutraceutical, and pharmaceutical properties [21–24]. Moreover, it was shown that fermentation improves protein digestibility and the bioavailability of minerals and other micronutrients [25].

The consumption of these beverages has the potential to reduce the adverse health and economic impacts of poor diets. Additionally, among the main benefits of cereal-based beverages, there is the possibility of consumption by vegetarians, vegans, and lactose-intolerant consumers [26]. This paper outlines the current achievements and further development for enhancing the functionality of the non-alcoholic fermented cereal beverage (NFCB) segment.

#### **2. Non-Alcoholic Fermented Cereal Beverage Segment**

#### *2.1. An Overview of NFCBs*

Reviewing the existent literature regarding some of the most studied NFCBs, it is shown that they are usually based on barley, maize, millet, oats, rice, rye, sorghum, and wheat [27,28]. Cereals are known as important sources of dietary proteins [29], energy, carbohydrates, vitamins, minerals, and fibre (arabinoxylan and β–glucan), but, at the same time, they are deficient in some basic components (essential amino acids, e.g., lysine) [19,30]. Whole grains are known to have important bioactive compounds and high nutritional values and their regular consumption has a positive effect on health [31–33]. The presence of soluble fibre lowers the glycaemic index of the beverage by slowing down its digestion and absorption, whereas phenolic compounds have antioxidant potential and scavenge harmful free radicals in the body [9].

Most of the traditional and currently produced NFCBs are considered functional foods and wholesome nutritional products [22,34,35]. Functional foods have been intensively studied in recent years and are continuously looked at as research efforts turn to processing food matrices, such as cereals, vegetables, and fruits, into medicine-like products.

A more detailed definition describes functional foods as industrially processed or natural foods that, when regularly consumed within a diverse diet at efficacious levels, have potentially positive effects on health outside basic nutrition [36]. This means that such products should provide a therapeutic benefit if consumed regularly within a diverse diet and with the condition of having the main nutrients extracted in a standardized manner and dosage [37].

Originally, cereal fermented beverages were mainly produced due to the need for conserving and utilizing various cereals and crops with affordable financial implications. Some of them, which are now commercially available as soft drinks and non-alcoholic beverages, were traditionally prepared as alcoholic beverages, having higher contents of alcohol [38].

Currently, "non-alcoholic" is a regulatory term and the laws regarding it vary across the globe. For example, EU regulation no. 1169/2011 simply states that a beverage must be labelled as an alcoholic drink if it has an alcoholic strength by volume (ABV) of over 1.2%. Moreover, in Great Britain, an alcohol-free drink has a maximum alcohol content of 0.05 % ABV; in Germany, a beverage is considered alcohol-free if the maximum alcohol limit is below 0.5 % ABV; in Spain, a non-alcoholic beer contains a maximum of 1 % ABV; in France, alcohol-free beers contain a maximum of 1.2 %; in US, non-alcoholic beers are of a maximum 0.5 % ABV; in China, non-alcoholic drinks can be of up to 0.5 % ABV [39]; in Japan, non-alcoholic beverages contain up to 1% ABV [40]. Depending on the country's traditional recipe, *boza*, a lactic acid fermented drink, has an alcohol content of less than 1% ABV in Turkey and up to 7% ABV in Egypt [41], probably due to microorganisms involved in the fermentation process [42].

Fermented drinks were and are obtained using a combination of fermentable substrates, like cereal mixes, fruits, plants, spices, legumes, and vegetables. The possibility of combining various fermentable substrates and of adding supplementary bioactive compounds to the final product encourages the development of different versions of the same original recipes, according to specific resources, health concerns, and nutritional needs. Such enhancements can be seen, for example, in vitamin A-fortified *mahewu* [43] or in establishing the suitable temperature for saccharification and oligosaccharide production efficiency in *amazake*, containing up to 0.5 % ABV [44].

Globally, there are numerous similar non-alcoholic cereal fermented beverages with similar names and profiles targeted for thirst quenching properties, on nutrition added value, cultural significance, and on providing alternatives to alcoholic beverages. Fermented beverages based on cereals are somewhat common around the world as staple foods, particularly in developing countries and are all made in a similar manner, generally using spontaneous microbial cultures [38]. Although there are various non-alcoholic cereal fermented beverages with different chemical profiles and sensory traits, all of them present certain bioactive compounds and therapeutic agents, which make them beneficial for human health. The functional compounds available and detected in NFCBs have variable values, therefore, it is difficult to assess their functional impact when consumed regularly [17]. If cereals lack certain nutrients, then additional food matrices can be added to enhance the final NFCB, as seen in a case of *mahewu* enhanced with *Moringa oleifera* leaf powder for elevating Ca and Fe contents [45]. Another example of a functional NFCB is a non-alcoholic beverage made of green tea and barley malt wort for delivering superior amino acid content [46].

In Table 1, there are several NFCBs listed with their place of origin, constituent cereals, microorganisms, nutritional compounds, and potential health benefits. As previously mentioned by other authors, the functional outcome of such products is strongly connected to the selection of the cereals, microorganisms, fermentation temperature and time, and other additional food matrices [47–49].

#### *Foods* **2020**, *9*, 1031

22

**Table 1.** *Cont.*

Some of the NFCBs presented in Table 1 are commercially available, with various scales of production, while others are mainly homemade beverages, produced for individual consumption using traditional methods. *Busa*, *kunun-zaki*, *mahewu*, *munkoyo*, *obushera*, *oshikundu*, *pozol*, and *tobwa* are produced in rural and urban areas and their production is essentially a home-based industry as for now, there is no large-scale factory production. *Amazake* is commercially available in Japan and it is classified as a soft drink [90,91]. *Bor¸s* is also industrially produced and used as a flavour enhancer in Romanian gastronomy or it is consumed as a refreshing drink [55]. *Boza* is one of the most popular Bulgarian beverages and is industrially produced at a large scale in all countries of the Balkan Peninsula [92]. *Kvass* is a traditional fermented Slavic and Baltic beverage and is one of the most popular beverages in Russia, numerous varieties emerged due to its popularity and market demand. Currently, *kvass* production is designed according to traditional processes, with the implementation of modern biotechnological methods [72]. *Shalgam* is a traditional beverage of southern Turkish cities and it is commercialized in various markets of European cities [86]. In Table 2, there are more details presented regarding NFCBs and their sensory properties, pH values, nature of use, fermentation status, and production scale.

**Table 2.** Non-alcoholic fermented cereal beverages and their sensory properties, nature of use, and fermentation status.

**Table 2.** *Cont.*

#### *2.2. Processing Technologies and Their Outcome*

The dietary attributes and sensory traits of cereal products can be at times viewed as inferior or deficient in comparison with those of milk and milk-based foods. Some of the reasons behind this include the smaller protein quantities, deficiency in certain amino acids (lysine), the presence of antinutrients (phytic acid, tannins, and polyphenols), and the coarse nature of grains [19].

The fermentation of starchy sources is more complex compared to that of low molecular sugars (glucose or sucrose) because, in general, the starch must at first be converted into fermentable sugars. To achieve an almost complete starch degradation, two main types of amylolytic enzymes are required (α-amylase and glucoamylase) [98].

Several methods have been engaged with the aim of enhancing the nutritional qualities of cereals. These include genetic improvement and amino acid supplementation with protein concentrates or other protein-rich sources, such as grain legumes or defatted oil seed meals of cereals. Additionally, several processing technologies, which include cooking, sprouting, milling, and fermentation, have been put into practice to improve the nutritional properties of cereals, although the best one is probably fermentation. In general, the spontaneous fermentation of cereals leads to a decrease in the level of carbohydrates, as well as some non-digestible poly- and oligosaccharides [35]. Certain amino acids may be synthesized, and the availability of B group vitamins may be improved [99]. Fermentation also provides optimum pH conditions for the enzymatic degradation of phytate, which is present in cereals in the form of complexes with polyvalent cations, such as iron, zinc, calcium, magnesium, and proteins. Such a reduction in phytate may result in a several fold increase in the amounts of soluble Fe, Zn, and Ca [19,100–102].

The current article advances one general processing technique, by compiling all the traditional recipes assessed and integrating germinated and non-germinated grains, to adapt it to an industrial scale with functional improvement. Several traditional processing steps can be applied in the production. The general outline of the process is essentially the same and that presented in Figure 1: the grains, after conditioning, are either soaked/wet milled or the grains are dry milled and the flour is extracted in water afterwards, the mix is boiled to gelatinize the starch, a source of enzymes to hydrolyse the gelatinized starch into fermentable sugars is added, and finally, spontaneous fermentation occurs [19,103].

**Figure 1.** General flow diagram of traditional NFCB production.

#### 2.2.1. Pre-Treatment of Raw Materials

Cereal processing is an essential component of the brewing production chain and the milling process is the main procedure. Before milling, the cleaning and conditioning of the grains is required. The cleaning process allows for the removal of various impurities, depending on the raw material types. The most common grain impurities are shrivelled grains, other cereals, grains damaged by pests, grains with discoloured germs, and sprouted grains. There are also miscellaneous impurities, including extraneous seeds, damaged grains, extraneous matter, husks, ergots, decayed grains, and insects. The conditioning or tempering of grains is performed using the monitored addition of water, which turns the endosperm softer and the bran harder. Doing this prevents the bran from breaking up, aids gradual separation throughout milling, and enhances sieving efficiency [104].

There are two milling categories, namely dry and wet milling, each having its own characteristics. Dry milling removes the germ and the outer fibrous materials of grains, as these by-products are not used in traditional ways [105]. Malting utilizes the power of natural germination when the grains, after absorbing water, germinate in the presence of oxygen to achieve a moisture content of up to 47% [106]. Another by-product is represented by floating kernels, which are unsuitable for malting. Throughout germination, the grain's embryo expands, and rooting begins. Moreover, the germination and steeping processes frequently overlap. It is recommended to keep the germination time and temperature at low values, given that long and warm germination processes lead to longer roots, resulting in larger malt yield losses [104,106].

During germination, grain enzymes start to break down the endosperm high-molecular-weight material into easily digestible components for the yeasts. Drying the malt stops the germination process [104].

In the case of traditional methods, the grains are usually superficially cleaned. A part of the cleaned grains (in variable percentages) is washed and soaked (10 to 20 h) at room temperature (26–35 ◦C). The soaked grains are drained and left for germination for 48 to 72 h with a frequent spraying of tap water. The germinated grains are sun dried (5 to 20 h), so the success of the process depends on the weather, mostly the intensity of the sunshine. Afterwards, the grains (malted and non-malted) are milled separately or in a mixture to obtain flour using rudimentary equipment [107].

Industrial cleaning processes aim at removing impurities and all other materials except for grains, using specific equipment such as magnetic separators, disc or sieve separators, aspirators, destoners, colour sorters, etc. The conditioning process ensures the complete hydration of grains, holding them in suitable containers for specific time intervals. Usually, depending on the grain varieties and initial moisture levels, the soaking time and temperature may be different [104].

For industrial-scale malting (Figure 2), the cereals are dried, and kilning is used to stop further transformations.

**Figure 2.** General flow diagram of industrial NFCB production (compilation from Encyclopaedia Britannica's brewing process).

During controlled drying, the water content should go under 5%, to stop the enzymatic activity whilst colour and flavour compounds are formed. A lot of by-products can additionally be recovered and further capitalized at an industrial scale. Dry milling can be extended to pearling, an abrasive procedure for gradually removing the testa and pericarp, aleurone and sub aleurone layers, and the germ. This results in polished grains and by-products with enhanced contents of bioactive compounds. Alternatively, wet milling, mostly used for producing starch and gluten, can increase the value of the cereals, as it is a source of by-products, with coproducts such as steep solids (abundant in compounds of pharmaceutical interest), germs (used in the oilseed-crushing industry), and bran [104].

#### 2.2.2. Mashing

The traditional mashing process differs greatly, depending on the local culture, and has low efficiency. In Africa, either sorghum or finger millet malt is commonly used as a source of enzymes [22,108]. In the case of gowé production, a beverage based on maize and sorghum, the grains are milled to flour, which is optionally mixed/kneaded with tap or hot water or with a supernatant of a previous production and left to ferment at room temperature. Additionally, this processing technique can include a saccharification step, where a part of the malted sorghum or maize flour is kneaded with water and left for saccharification for 5 to 10 h [107]. For munkoyo, a maize-based beverage with a variable ABV, the specific feature is the usage of *Rhynchosia* roots or watery root extract as an enzymes source [22,77,78]. Starch gelatinization facilitates the activity of α- and β-amylases from the *Rhynchosia* roots for at least 4 h up to a maximum of 24 h, which hydrolyse the starch into fermentable sugars [22,103,109]. Mahewu is an example of a non-alcoholic sour beverage made from corn meal, consumed in Africa and some Arabian Gulf countries [74]. It is prepared from maize porridge further mixed with water. Sorghum, millet malt, or wheat flour is then added and left to ferment [19]. The production techniques of obtaining kvass, a cereal-based beverage produced from rye and barley malt, rye flour, and stale rye bread, uses as raw material either stale sourdough bread or malt rye malt and rye flour [110]. Boza is a colloid suspension, non-alcoholic beverage consumed in Bulgaria, Albania, Turkey, and Romania, made from wheat, rye, millet, maize, and other cereals mixed with sugar or saccharine [92]. Boza's preparation involves six stages: the preparation of raw materials, boiling, cooling, straining, the addition of sugar, and fermentation. Another option for its production is the use of previously fermented boza as an inoculum [59].

The industrial mashing process resembles the beer production process. Between one and three volumes of water are added per volume of milled grains, and during the cooking process, the mixture turns into a mash. The mixture is cooked at a normal pressure or in an autoclave for about 2 h at 4–5 atmospheres.

The macromolecular profile of cereal-based beverages is generally determined by polymeric compounds (proteins, polysaccharides, and polyphenols) and their progress in depolymerization during processing [111,112]. Given that yeasts or specific strains of lactic acid bacteria (LAB) cannot metabolize high-molar-mass substances [113], the macromolecules are solely depolymerized during the malting and mashing process by the malt's intrinsic enzymes. During malting, the enzymatic degradation of polymers is technologically controlled by the degree of steeping, germination time, and germination temperature [114,115]. Modern brewing barley varieties are bred to be balanced in malting performance and to meet the required brewing specifications. The degradation of starch into fermentable sugars (amylolysis) is the primary objective of mashing (substrate production for fermentation) [116], since during the subsequent fermentation process, only low-molar-mass compounds, fermentable sugars, and low-molar-mass proteinaceous compounds are metabolized by microorganisms (e.g., LAB and yeasts) [117,118].

Lautering is the next step in the large-scale process, through which solid and liquid fractions, respectively, the spent grain, composed of sugar-extracted grist or solids remaining in the mash, and the sweet wort with high contents of fermentable sugars are obtained. The spent grain is the major by-product of the brewing industry and represents a valuable source of bioactive ingredients and a potential ingredient for functional foods [119]. In small-scale production, malt extract can be used instead, thus skipping the use of grain malt (including milling, mashing, and lautering) [120].

#### 2.2.3. Cooling and Addition of Yeast, LAB Cultures, and Other Ingredients

After boiling, the mash is gradually mixed with cold water in a 1:1 ratio. This slurry is often filtered or decanted to remove the grinding waste and insoluble plant material. In many traditional processes, where cereals are soaked in water for a few days, a succession of naturally occurring microorganisms will result in a population dominated by LAB. In such types of fermentations of endogenous grains, amylases generate fermentable sugars, which serve as energy sources for lactic acid bacteria. When no malted cereals are used, sucrose is added to the beverage to mimic the malt's sweet taste.

The bacterial flora formed in each fermented cereal drink is influenced by several factors, such as water activity, pH level, salt concentration, temperature, and the composition of the grain matrix, which must be considered in industrial processes. However, most fermented drinks, including the well-known products commonly met in the Western world, as well as those beverages of other origins which are less well studied and characterised, rely on lactic acid bacteria to mediate the fermentation process [19,121]. Lactic acid fermentation contributes towards the nutritional value, shelf life, safety status, and acceptability of a wide range of cereal-based foods [118]. Fermentation is often just one step in the process of fermented food preparation. Other operations, such as volume reduction, salting, or heating, also affect the final product characteristics [19,122]. Depending on the desired product, further steps can be applied afterwards, such as standardisations and the addition of other ingredients like flavourings, sugar, and stabilizers.

#### 2.2.4. Fermentation Process

Despite the lack of process control, dealing with unstandardised microbial flora composition, delayed fermentation, and imperfect reproducibility of the fermentation process, spontaneous fermentation offers complex microbial diversity, providing higher levels of intrinsic stability to the microbial community [123]. This is achievable due to stabilizing interactions between species that prevent and inhibit the proliferation of unwanted microorganisms, including pathogenic ones. Recently, the existence of stability criteria for complex microbial communities was proven [124]. In terms of spontaneous fermentation, this can be explained by the resilience to small perturbations when there is a balance between the availability of resources, namely nutrients, and consumers (e.g., lactic acid bacteria). Commonly, the production of artisanal fermented beverages is conducted in successive batches [55], by using a natural starter from the previous fermentation. At all traditional sites, spontaneous fermentation proceeds after cooling. A 24 h fermentation period is sufficient for

some traditional beverages to develop their characteristic sensory attributes, although in practice, fermentation time can go on for up to three days. Some brews, obtained through a 6 to 15 h fermentation interval of non-malted grain flour, are enhanced with commercial sugar (sucrose) to obtain the sweet taste. This process is a distortion of the original germination technique for gowé production [22]. A modern industrial process is different from the traditional process regarding the introduction and control of thermophilic LAB cultures, which only produce lactic acid, the extension of the product's shelf life by pasteurization and/or chemical preservation, and the inclusion of sugar and/or artificial sweeteners [125,126]. Controlled fermentation also leads to a general improvement in the shelf life, texture, taste, and aroma of the final product. During cereal fermentation, several volatile compounds are formed, which contribute to a complex blend of flavours [127]. Moreover, there is a good opportunity to apply colloidal dispersions in the form of nanoemulsions to deliver food grade nanoparticles, which contain water-insoluble molecules that were formerly unsuitable due to their poor soluble characteristics. Thus, there is a wide range of healthy foods which can be further designed, such as cereal-based fermented beverages enhanced with nanomolecules possessing beneficial health attributes [27].

#### *2.3. Fermentation Microbiota and Safety of NFCBs*

Originally, fermented beverages were only consumed in their native regions, however, due to increasing demand and interest, some of those traditional beverages are available to international markets. The attributes of traditional fermented beverages are influenced by several factors, such as the use of different raw materials, manufacturing methods, natural microbiota, and fermentation conditions. The microbiology of many traditional fermented drinks prepared from the most common types of cereals is quite complex.

There is a lot of diversity in the traditional processing techniques used for cereal-based fermented beverages all over the world, integrating single or multigrain cereals, germinated and/or non-germinated grains. Many types of cereal-based fermented beverages are produced in Africa, such as *togwa* in Tanzania, *mahewu* in Zimbabwe and South Africa, *maw*è in Benin, and *munkoyo* in Zambia and the Democratic Republic of the Congo [9,19,128,129]. Generally, the preparation of these products is a traditional family activity with an uncontrolled fermentation process by diverse microbial communities. The composition of the microbial community in a fermented food product largely determines the key product properties [130,131]. In other words, variations in microbial communities may result in differences in product quality, taste, acceptability, and microbial stability.

In one of our previous papers [55], we proved the impact of processing parameters, namely temperature and batch fermentation cycles, on the chemical composition of bors, . Interestingly, the final composition of cereal-based beverages might not only be influenced by the physical process parameters. Processing practice variation affects the microbial composition of the fermenting microbial community. Despite the decreased pH caused by the lactic acid bacteria activity, the low pH of munkoyo also permitted the development of acidifying bacteria [22]. An important role is played by the initial microbial composition of the raw materials used in the process. The same study also referred to the fact that further investigations might be needed into the soil composition of the harvested raw materials. Along with the prevention of growth of most pathogenic strains [132], a low pH (of 2.5 to 3.5) improves food safety and expands the shelf life of this type of beverage [22]. The pathogenic microorganisms that are aerobes and facultative anaerobes and ferment simple sugars have an optimum pH for growth of 6.0 to 8.0. However, growth can occur at a pH as low as 4.3 and as high as 9, but with a combination of factors (pH, water activity), the control of foodborne pathogen growth can be done [133].

The fermentation of most cereals is natural and involves mixed cultures of yeasts, bacteria, and fungi [134]. Some microorganisms may participate in parallel, while others act in a sequential manner with a changing dominant flora during fermentation. The challenge, though, is the generally uncontrolled nature of the fermentation, which raises safety concerns, as well as the lack of standardization in the methods used, thus further research and development are needed to

improve the traditional fermentation processes [80]. In this regard, the introduction of starter culture technology has led to greater consistency and safety and to better product quality [135].

Furthermore, the main difficulties encountered in the production of cereal-based beverages using traditional processes are linked to the high variability of unit operations and the unhygienic conditions of the processing environment. The soaking and germination parameters (temperature, duration, moisture) vary within and between processors. Moreover, during soaking and germination, the grains can be infested by fungi with the potential development of mycotoxins (aflatoxins) [136].

The continuous study of the fungi, yeasts, and bacteria strongly involved in ensuring a certain quality of the NFCB allows for the optimization of same-product delivery [85,137,138]. Such upgraded fermented beverages are sometimes the outcome of general efforts to enhance original recipes [139]. *Kunun-zaki* is an example of a traditional wheat and sorghum fermented beverage now also commercially available in the form of a powder [68], and there are several strategies proposed to upgrade and re-engineer the process of *gowé* production, a beverage obtained from sorghum and maize [66]. As seen so far, it is possible to prolong shelf life through the co-incubation of probiotic cultures, as seen in the development of some cereal-based fermented beverages [37,140] or to market to a group of consumers looking for healthy and functional foods by using oats, for example, as a main ingredient [141]. In-depth studies are still being conducted to ensure food safety in the processing technology of fermented foods through the keen selection of starter cultures and thorough examination of the specific microorganisms [142,143]. For example, it was recently shown that *Enterococcus faecium* YT52 isolated from *boza* is susceptible to clinically relevant antibiotics and contains low numbers of virulence factors and antibiotic resistance genes. Therefore, the enterocin-producing *E. faecium* YT52 strain poses a low risk to consumer health, and this strain may be used as a starter or a co-starter culture for improving the food safety of fermented products by acting against foodborne pathogens, such as *Listeria monocytogenes* and *Bacillus cereus* [144]. Moreover, rethinking the technological processes for obtaining various cereal-based fermented beverages helps to increase their functionality and overall therapeutic and nutritional properties. Such an example is *boza*, enhanced by fermenting cereals with *Lactobacillus acidophilus*, *Bifidobacterium bifidum*, and *Saccharomyces boulardii*, and by adding chickpea flour to the fermentable substrate for an elevated protein content [145]. Along with these interventions, narrowing the risks of product spoilage through the inoculation of specific strains of LAB and yeasts and processing the cereals in a certain manner repetitively allows the producers to deliver same-quality functional NFCBs.

Fermentation is one of the oldest known biotechnologies and the most economical procedure for producing new foods and ensuring product conservation. The yeasts responsible for fermentation in fermented drinks include species of *Saccharomyces*, *Saccharomycopsis*, *Schizosaccharomyces*, *Pichia*, *Candida*, *Torulopsis*, and *Zygosaccharomyces*. Brewer's yeast, *Saccharomyces cerevisiae,* metabolizes various sugars, mainly into alcohol, but also into other flavour-active substances. The most used microorganisms in fermentation processes belong to *Lactobacillus* species, which synthesize many flavour-active substances and lactic acid and are considered probiotic microorganisms known to support intestinal microbiota [146]. Fermented beverages are known to be rich in bioactive compounds, such as immune globulin peptides and the bioactive hormone cytokinin [19,71,82,147,148]. The use of a wide variety of microorganisms and yeasts is implied in the production of NFCBs by both traditional and modern means.

Largely responsible for the fermentation process are the indigenous microbiota present on the substrate or which can be added artificially as a culture. Basically, there are four main fermentation processes, which include alcohol production, lactic acid development, acetic acid production, and alkaline fermentation [19]. The modern industry for fermentative foods and beverages is innovative, given that it currently employs thermophilic fermentation, DNA technologies, molecular devices, designed starter cultures, genetic engineering, etc. Recombinant DNA technology has provided new insights for enhancing the product quality by designing tailor-made starter cultures that perform better than those found naturally [60]. For example, Basinskiene et al. used *Lactobacillus sakei* KTU05-6 and *Pediococcus pentosaceus* KTU05-10 to ferment extruded rye for obtaining *kvass*, a traditional Lithuanian NFCB. They showed that the pH of the beverages fermented by LAB reached lower values compared to yeast fermentation and a they had a higher amount of organic acids. Innovative technology was applied, such as xylanolytic enzymes and antimicrobial active LAB, to improve the product's functional properties [71].

Functional cereal fermented beverages are becoming more attractive as they represent healthy alternatives for lactose intolerant consumers and for those who avoid certain allergens. For example, a study describes the production of kefir-like riboflavin-enriched beverages based on oat, maize, and barley flours. To obtain this beverage, riboflavin-producing Andean LAB were used, consisting of five *Lactobacillus plantarum* strains and two *Leuconostoc mesenteroides* strains [149].

As previously discussed, ensuring product safety, although difficult at times, is a crucial step in the production of traditional beverages where the fermentation process is spontaneous. Thus, microorganisms found on the brewers' skin, hair, and clothes can alter the product safety, therefore, high standards of hygiene are mandatory. Nevertheless, through the identification of bacteria strains and yeasts, the safety and quality status of the fermented beverages are guaranteed. A safety case study was conducted concerning "*obushera*", a Ugandan traditional fermented cereal beverage where important steps, such as pasteurization and ensuring water quality, are in the loop to ensure product safety and quality, as pathogens can also change the product's sensory characteristics [80].

Superior results can be obtained in the production of NFCBs by better understanding the interaction of microorganisms in the fermented substrates. The beneficial outcomes of controlling microorganisms are mirrored in product safety, increased shelf life, improved nutrient contents and availability, palatability, and enhanced sensory traits. For example, it was shown that *S. cerevisiae* improves LAB growth by transmitting essential metabolites, such as pyruvate, amino acids, and vitamins, while it uses some metabolites produced by LAB as carbon sources [150]. Moreover, Salari et al. concluded in their study that malt and *L. delbrueckii* were the best substrate and lactic strain for producing a functional beverage with the highest cell viability (1.2 × 106 cfu/mL after 4 weeks) [151].

#### Probiotics

Non-alcoholic fermented cereal-based beverages contain a wide range of diverse probiotics, depending on their cereal substrate and overall production methods. The processing method should assure the stability of the bacterial composition in order for the final product to possess probiotic functionality [152]. Still, the threshold to declare a beverage a probiotic one must be higher than 107 CFU/mL. Moreover, not all lactic acid bacteria possess probiotic activity. *L. rhamnosus*, also present in NFCBs (Table 1), was efficient in treatment and prevention of gastrointestinal disease [152]. The traditional Romanian NFCB, *bors,* , has been consumed since ancient times as a gastric remedy. Several potentially probiotic bacteria—*L. casei*, *L. plantarum*, *L. brevis*, and *L. fermentum*—were isolated from bors, , explaining the traditional consumption of this beverage for curative purposes [56].

The predominant microorganisms in the spontaneous fermentation of the African *mahewu*, a non-alcoholic sour beverage made from corn meal and consumed in Africa and some Arabian Gulf countries, belong to *Lactococcus lactis* subsp. *lactis*. On the other hand, the microbiota identification of Bulgarian boza shows that it mainly consists of lactic acid bacteria and yeasts, such as *Lactobacillus plantarum*, *L. acidophilus*, *L. fermentum*, *L. coprophilus*, *Leuconostoc ra*ffi*nolactis*, *Ln. mesenteroides*, and *Ln. brevis* and *Saccharomyces cerevisiae*, *Candida tropicalis*, *C. glabrata*, *Geotrichum penicillatum*, and *G. candidum*, respectively [19].

In the case of Turkish *boza*, the use of LAB and yeast isolates as starter cultures allows for controlled fermentation studies to be carried out. The selection of proper strains with probiotic and antimicrobial properties enhances the functional properties of *boza* [59].

As seen in the case of African countries, the primary challenge for the development and use of fermented cereal-based probiotic beverages is the common lack of knowledge regarding the health and nutritional benefits of such foods and beverages. Insufficient common knowledge on probiotics and their benefits creates a sense of scepticism among consumers. Moreover, there is also an imperative need to ensure proper facilities for probiotic starter cultures, given that through spontaneous fermentation, the organoleptic and functional qualities of the resulting products are variable [153].

#### *2.4. The Nutritional and Bioactive Composition of Commonly Consumed NFCBs*

Consumers are aware of the importance of maintaining a strong immune system to prevent illnesses and they are actively looking for products which can help maintain their health status and alleviate health problems. It has been scientifically proven that probiotics isolated from functional beverages boost the immune system [154], and that, along with prebiotics, they are able to improve the intestinal homeostasis, immunomodulating ability, and general health of the host [27].

Looking at past publications, it is shown how fermented beverages have transitioned from traditional natural fermented products to beverages formulated with functional ingredients meant to offer cardiovascular health benefits, and then to functional fermented drinks which improve gastrointestinal health, which could then evolve into fermented products containing specific bioactive nanoparticles [134].

NFCBs are receiving increased attention from researchers and consumers more recently due to their proven probiotic characteristics and disease prevention perspectives [17]. The perceived health outcomes of fermented beverages are strongly related to the microbial content and implicit improvement of gastrointestinal health [38]. Moreover, non-alcoholic fermented beverages offer a sense of wellbeing, as they stimulate the metabolic system [126].

Fermented cereal-based foods, including NFCBs, are a potential source of new functional lactic acid bacteria species besides various nutrients and bioactive compounds, with beneficial effects on human health [56]. Furthermore, as briefly mentioned previously, NFCBs are healthy alternatives to the traditionally consumed food probiotics of dairy origin (e.g., yogurt, kefir, etc.), especially for people with lactose intolerance and milk protein allergies [27].

Given the functional components of fermented beverages and their bioactive compounds released through fermentation by cultures, NFCBs have been linked with many potential and some proven health benefits and actions on digestive, endocrine, cardiovascular, immune, and nervous levels [155]. They present beneficial actions for vital body functions and contribute to the prevention and reduction of risk factors for various diseases [19,23]. NFCBs are rich sources of minerals, vitamins, fibre, flavonoids, phenolic compounds, antioxidants, omega-3 fatty acids, plant extracts, sterols/stanols, amino acids, and biopeptides, among others, which could also protect from oxidative stress and inflammation diseases [17,134]. The presence of numerous valuable compounds in NFCBs grants several health benefits upon consumption, as presented in Figure 3.

**Figure 3.** Health benefits associated with NFCB consumption.

Considering group B vitamins, these are unequally distributed in grain tissues. What counts the most in terms of their potential functionality in cereal-based food and beverages is their biological availability. During thermal food processing, these vitamins are destroyed almost completely, however, lactic acid fermentation represents a great tool for food industrialists interested in developing novel vitamin-fortified products. For example, Capozzi et al. (2012) pointed out the ability of many lactic acid bacteria strains, such as *Lactobacillus delbrueckii, Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus fermentum*, and *Pediococcus lactis*, reported in the composition of several NFCBs (Table 1), to biosynthesize riboflavin [156].

A new concept attributed to non-alcoholic cereal-based beverages is that they are healthy drinks with important impacts on human health. It has been shown that consuming NFCBs leads to liver function improvement, increased levels of lactobacilli and bifidobacteria in the intestinal microbiota [157], balanced gut microbiota, and the prevention of bacterial translocation with reduced incidences of nosocomial infections [27]. Furthermore, because non-alcoholic fermented beverages contain several LAB metabolites, their consumption confers bactericidal, bacteriolytic, and bacteriostatic properties, resulting in therapeutic effects at a digestive level. Antimicrobial compounds identified in functional beverages exhibited activities against several Gram-positive and Gram-negative bacteria and against yeasts and moulds [17]. The functional compounds and potential or proven health benefits of NFCBs are presented in Table 1.

As previously discussed, the potential benefits of NFCBs can be ensured in a safe and non-contaminated production environment to prevent the risk of product spoilage. Moreover, there are diverse nutritional and bioactive components of NFCBs, depending on the cereal substrate, microorganisms, and fermentation parameters, and on the technological process followed.

Currently, novel NFCBs are designed based on traditional recipes for delivering improved functional fermented beverages. Such an example is "*amahewu*" or "*mahewu*", a southern African LAB-fermented non-alcoholic maize-based beverage, which is deficient in vitamin A. In a study published in 2015, the traditional drink was redesigned using provitamin A-biofortified maize in exchange for the traditionally used white maize and it resulted in a functional beverage addressing vitamin A deficiency, a concerning health problem in sub-Saharan Africa [43].

#### 2.4.1. Phenolic Compounds and Antioxidant Activity

Cereal grains and germs contain various major phytochemicals, such as phenolic acids, flavones, phytic acid, flavonoids, coumarins, and terpenes. The bran layers of cereal grains are relatively rich in water soluble and liposoluble antioxidants. Oats include phenolic acids, flavonoids, tocopherols, tocotrienols, and avenanthramides. On the other hand, insoluble fibre of wheat bran contains about 0.5%–1% phenolics and most phenolic compounds, like ferulic acid, are abundant in whole grains [158].

Depending on the cereal substrate, fermentation type, and other added ingredients, each NFCB can have quite different compositions. For example, the phenolic profile of brown beer vinegar indicates that such a fermented beverage is a rich source of phenolic compounds (661 mg GAE/L) and antioxidants, with 30% increased antioxidant activity after acetic fermentation. The same trend was observed regarding some of the drink's individual phenolic compounds. With cohumulone I (4.44 mg CE/L), cohumulone II (6.58 mg/L), 8-prenylnaringenin (2.33 mg CE/L), 6- prenylnaringenin (1.86 CE/L), humulone (5.62 CE/L), and isohumulone (4.14 CE/L) [159], this non-alcoholic fermented beverage can be considered an alternative source of valuable compounds, which could also be part of specific diets.

Fermentation parameters are of high importance in what concerns an NFCB's antioxidant profile. For example, a fermentation temperature of 24◦ C was considered a positive factor influencing the antioxidant activity of *bors,* , a Romanian traditional wheat bran-based fermented beverage [55]. The antioxidant activity ranged from 3.05 to 8.52 mmol/L Trolox.

Some of the ancient traditional fermented beverages are currently produced at industrial scale. Still, the original recipes and the nutritional components are being altered because of thermal treatments

applied to obtain product safety and stability. The addition of natural starters allowed for the increase in phenolic contents and the enhancement of final antioxidant activity. The same trend considering phenolic content between fermentation stages was found in the case of *bors,* [56]. Depending on the processing operations applied, the phenolic compounds ranged considerably (4-hydroxybenzoic acid: 0.9–5.9 mg/L; vanillic acid: 0.6–3.2 mg/L; syringic acid: 0.5–2.5 mg/L; p–coumaric acid: 0.5–1.5 mg/L; sinapic acid: 0.6–2.7 mg/L; ferulic acid: 13.7–47.8 mg/L).

#### 2.4.2. Amino Acids

Although cereals pose few challenges from a nutritional standpoint, especially with regard to the increased starch content upon cooking, the limited amino acid contents of their protein fractions, or the reduced bioavailability of their zinc and iron contents, there are already some solutions meant to correct and further enhance the nutritional status of cereal-based foods and beverages [160].

For example, in a study conducted on *bors,* , an increase in amino acid content was found from one fermentation stage to another. The same study stated that the LAB fermentation of cereals improves the protein quality as well as the level of certain free amino acids by enhanced endogenous proteolysis and/or microbial action. Among the identified amino acids, there were isoleucine and threonine [56].

In general, the natural fermentation of cereals allows for amino acids to be synthesised, while it also helps to enhance the availability of group B vitamins. Fermentation processes also offer suitable pH conditions for phytate enzymatic degradation. Such a reduction in phytate may increase the amount of soluble iron, zinc, and calcium several fold, correcting the nutritional status of cereal-based beverages and foods [19].

#### **3. Future Perspectives to Enhance the Functional Properties of NFCBs**

#### *3.1. Consumers' Preferences and Requirements*

The way we perceive healthiness in foods varies between cultures and is reflected in the consumers' familiarity with health-related information [161]. Moreover, modern-day buyers might show increased interest in foods which can improve well-being [162], reduce the risk of developing illnesses, and satisfy nutrition-related conditions such as food intolerances and allergies. Currently, consumers approach the concept of wellness with a holistic view and are becoming more health conscious, particularly due to the growing incidence of diseases such as type 2 diabetes, coronary heart disease, cancer, and obesity [163]. There is a heightened demand for food products which are nutritious, functional, attractive, with clean labels, and which are ready to eat. Considering such attributes, the beverage sector seems to be one of the most suitable for addressing customers' demands and for increasing the rate of functional product consumption. Market data indicate that functional food products are among people's choices even when economic issues arise. The global functional beverage market has increased rapidly, and it is expected to grow further, due to their sensory appeal and health-promoting attributes [38].

Studies showed that providing information on labels regarding health-related claims associated with functional beverages determines consumers' preferences for a specific product [164]. Not only health-related claims but also the sensory properties determine consumers' preferences for a specific functional beverage [34]. It was suggested that the importance of specific testing conditions for functional beverages will help product developers to reformulate the product with the proper design of experiments, focusing on the consumers' needs [165], as they will determine the product acceptance [166]. The urgent need for educational campaigns designed with accessible and easily understood wording about the nutritional components of foods has already been pointed out [167]. Although the previously mentioned study was conducted in Brazil, insufficient knowledge about nutritional facts for food products is a worldwide issue strongly related to the level of education [168].

#### *3.2. Possibilities of Improving the Appeal and Functionality of NFCBs*

The functional beverage market is competitive and driven by product innovation and health awareness trends concerning optimum nutritional diets. The biological activities and the sensory properties of a beverage arise from individual components, along with chemical and physical interactions within the food matrix during processing, storage, ingestion, and digestion [169]. Beverages deliver nutrients, bioactive components, antioxidants, vitamins, minerals, fatty acids, plant extracts, probiotics, prebiotics, and micronutrients [13]. Cereal-based beverages include a complex mix of different polymers, such as proteins, polyphenols, and polysaccharides. These polymers affect the sensory perception of beverages in terms of mouthfeel, depending on their substance properties [112].

Several strategies have been set to intensify the production, availability, accessibility, and consumption of beverages rich in bioactive compounds. These include combining cereals with pseudocereals (e.g., quinoa, amaranth, etc.), legumes, vegetables extracts, fruits, aromatic plants, and herbs to improve the quality of the final food product [19,56,170,171]. Furthermore, mixing cereals with legumes could improve protein quality [172]. Fruit juices (e.g., apple, pineapple, mango, orange, lemon, peach, lychee, and strawberry) are considered as health-promoting foods and are an important basis of enrichment on which to append an extra functional constituent that can significantly augment the appeal to customers. Cereal beverages are based on grain suspensions. The viscosity, mouthfeel, and sweetness of the drink can be adjusted to the consumers' tastes using enzyme compounds. Wort can be mixed with various plant- and fruit-based juices to obtain a beverage rich in dietary fibre suitable even for athletes and sport amateurs. Moreover, innovative flavours can be obtained by fermenting the extract with specific microorganisms. As previously mentioned, fruits were also employed in the overall production scheme of fermented cereal beverages, as these are the added sugar necessary for initiating the fermentation process. Chemical analyses of ancient organics absorbed into pottery jars from the early Neolithic village of Jiahu, Henan Province, China have revealed that a mixed fermented beverage of rice, honey, and fruit (hawthorn fruit and or grape) was being produced as early as the seventh millennium B.C. [173]. Such findings inspire the development of new functional products, such as yogurt-like beverages made of a mixture of rice, barley, emmer wheat, oats, soy, and grape must [174] or of other milk-based functional drinks using fermented plant juices [175,176].

Plants are valued for their nutrients such as vitamins, dietary fibre, antioxidants, and flavonoids, which have shown nutritious and health-promoting properties [134]. Bioactive compounds derived from fruits and vegetables can be good vehicles for probiotics, prebiotics, and synbiotics (e.g., fermented cereal beverages) [57,58]. Spices such as tarhana herb, mint, and thyme are mixed with wheat flour, yogurt, vegetables, and herbs to produce a traditional Turkish fermented food called tarhana [177,178] and improve taste, aroma, and other profile characteristics. Moreover, it is suggested that adding herbs with proven beneficial compounds to otherwise traditional fermented soft drinks can augment the nutritional profile of fermented cereal beverages and add therapeutic potential [179]. Among the suggested herbs, we can find echinacea (*Echinacea angustifolia*) for antibiotic action and immune system support, ginkgo (*Ginkgo biloba*) for enhancing memory and alertness, guarana (*Paulina cupana*) for improved cognitive performance, kava (*Piper methysticum*) for stress reduction and mental balance, and St John's Wort (*Hypericum perforatum*) for anxiety reduction [180]. Vegetables such as beets, tomatoes, carrots, and cabbage can also be included in the production of some functional NFCBs, as they provide supplementary fermentable substrates and nutrients, and act as prebiotics in the final product [181]. *Shalgam* is a traditional Turkish NFCB made of turnip bulb, purple carrot, salt, sourdough, bulgur, or bulgur flour [182], and is thought to regulate the digestive system's pH and to act as an antiseptic agent. The results of a research study conducted on the co-culture probiotic fermentation of a protein-enriched cereal medium suggests that plant protein may be exploited for achieving protein supplementation of NFCBs. Legumes like chickpeas, individually or in combination with cereals, can provide a good substrate for probiotic microorganism production [145]. Additionally, the composition of legumes, vegetables, and fruits is known to be rich in protein, phytochemicals, dietary fibre, vitamins, and other micronutrients beneficial for human health [183].

Currently, concepts like "green consumerism" and "minimally processed foods" are on the rise, as consumers prefer food lacking synthetic additives. Natural compounds that can be added to functional beverages, such as essential oils and plant extracts including rosemary, peppermint, bay, basil, tea tree, celery seed, and fennel with antimicrobial activity, might represent an alternative to synthetic preservatives. The challenge is to define the optimal dosage of such compounds to have a positive impact on the product's final nutritional status, sensory properties, and consumer acceptance [12].

#### *3.3. Perspectives for Future NFCBs*

Due to the increasing prevalence of lactose intolerance and milk protein allergies, as well as the general aim of controlling cholesterol intake, and along with the growing interest in vegetarianism, research efforts have been focused on the feasibility of using cereals as fermentation substrates for the development of probiotic, prebiotic, and synbiotic beverages [27,113,184]. In the production of various non-alcoholic drinks, probiotic lactic acid bacteria are used to boost the product's functional value [143]. Although ensuring nutritive compounds within NFCB is a crucial step for product development, flavour improvement remains one of the main challenges for the development of LAB-fermented beverages obtained from cereal-based substrates [185].

It is a fact that improved and more appealing NFCB sensory properties would help consumers shift their interest intensively towards such healthy products. The efficacy of probiotics in the treatment of bowel disorders, the prevention of antibiotic-associated diarrhoea, and the improvement of lactose metabolism has been proven [186]. It has been also concluded that both fermentation and acidification with lactic acid have the potential to improve the nutritional quality of cereal-based foods as a method to combat protein malnutrition and iron and zinc deficiencies [187].

Biotechnological processes, such as malting and lactic acid fermentation, are recommended for producing functional beverages with increased contents of functional bioactive components [188]. There are numerous important benefits of enhancing NFCBs through biotechnological processes and it is worth mentioning the reduction of phytates through LAB-fermentation, which in turn leads to the increased absorption of Fe and other minerals [189]. Due to cereal fermentation, quantitative and qualitative changes take place in small molecules, ensuring the high bioavailability of macro- and micronutrients [190,191]. As mentioned earlier, prebiotics and dietary fibre can be added to fermented drinks to heighten their nutritive and functional contents. For example, it was shown that the addition of soy fibre not only improves *Lactococcus lactis* counts but also enhances the beverage characteristics regarding acidity, viscosity, and syneresis [192].

Currently, emerging techniques, such as nanotechnology, are in discussion to enhance the nutrient composition and functionality of NFCBs. However, continuous research and technological improvements are required to better design NFCBs and ensure product safety. It is also crucial to improve the quality of the main ingredients and it is imperative to integrate food safety management systems for industrial scale production. The proposed solutions include the development of new production technologies for obtaining functional NFCBs by extending the spectrum of raw materials used and applying new biotechnological resources (enzymes and lactic acid bacteria) [71].

#### **4. Conclusions**

Science and technology have the potential to produce superior functional NFCBs and to deliver consistent product quality, to improve shelf life, and to enhance nutritional values to finally meet the consumers' demands. Past research studies have proven that lactic [55] and acetic [159] fermentation enhance the nutrient content and their bioaccessibility in the case of cereal-based fermented beverages. Adding probiotics and prebiotics to a beverage is perhaps one of the most convenient ways of turning it into a functional beverage.

The research efforts on the enrichment of non-alcoholic fermented cereal beverages are still in their early stages but appear to be more promising than ever. Upcoming studies should focus on traditional non-alcoholic fermented beverages around the world to identify new potential probiotic microorganisms and starter cultures, new ingredients as potential substrates, and to elucidate the contributions of microorganisms and enzymes to the fermentation process.

Knowledge about processing applications and bacterial strains is essential to control the production methods and to design proper mixes of microorganisms, ensuring product safety. Afterwards, starter cultures with expected outcomes can be used for the industrial production of standard-quality fermented beverages with functional attributes.

The effect of advanced technologies on NFCB functional properties during processing requires additional studies to ensure that these technologies can prevent the loss of product quality and nutritive compounds. Moreover, for both scientific and industrial actors, the main challenge is to manage the large-scale production of fermented beverages without losing the unique flavours and other properties associated with the original products. Given this, it is highly recommended to explore the sensory properties of NFCBs when obtained from a combination of cereals, legumes, fruits, and plants.

The current review investigates some of the most scientifically documented traditional non-alcoholic beverages from all over the globe, highlighting their functional compounds and associated health benefits upon consumption. Moreover, processing technologies and their outcome were highlighted and discussed from a large-scale production standpoint. NFCBs were also reviewed, concerning fermentation microbiota and product safety, highlighting the need to apply starter cultures to ensure food safety and standard quality. The nutritional and bioactive compositions of commonly consumed NFCBs were reviewed, showing the functional compounds of NFCBs and their associated health benefits.

NFCBs are associated with functional benefits to one's health status, as discussed. However, new studies should be carried out to produce new NFCBs, combining probiotic fermented beverages and products such as fruit juices, vegetables, and cereals, meant to address specific health concerns. Future cereal-based fermented beverages need to be balanced regarding sensory properties, nutritional composition, alcohol content, and resource investments to be even more attractive, healthy, and affordable.

**Author Contributions:** Conceptualization, L.C.S. and M.V.I.; Writing—original draft preparation, M.V.I., L.C.S., C.R.P., O.L.P., T.E.C. and A.B.; Writing—review and editing, L.C.S., M.V.I., A.P. and T.E.C.; Supervision, L.C.S., M.T., A.P. and E.M.; Funding acquisition, L.C.S. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by two grants, as follows: from the Ministry of Research and Innovation, CNCS-UEFISCDI, project number PN-III-P2-2.1-CI-2018-1462, within PNCDI III; and from the European Foundation for Alcohol Research (ERAB), Belgium, project number Ref EA 15 45.

**Acknowledgments:** The publication was supported by funds from the National Research Development Projects to finance excellence (PFE)-37/2018–2020 granted by the Romanian Ministry of Research and Innovation.

**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/).

### **Nutritional Features and Bread-Making Performance of Wholewheat: Does the Milling System Matter?**

### **Maria Ambrogina Pagani 1, Debora Giordano 2, Gaetano Cardone 1, Antonella Pasqualone 3, Maria Cristina Casiraghi 1, Daniela Erba 1, Massimo Blandino 2,\* and Alessandra Marti 1,\***

Received: 27 June 2020; Accepted: 29 July 2020; Published: 1 August 2020

**Abstract:** Despite the interest in stone-milling, there is no information on the potential advantages of using the resultant wholegrain flour (WF) in bread-making. Consequently, nutritional and technological properties of WFs obtained by both stone- (SWF) and roller-milling (RWF) were assessed on four wheat samples, differing in grain hardness and pigment richness. Regardless of the type of wheat, stone-milling led to WFs with a high number of particles ranging in size from 315 to 710 μm), whereas RWFs showed a bimodal distribution with large (>1000 μm) and fine (<250 μm) particles. On average, the milling system did not affect the proximate composition and the bioactive features of WFs. The gluten aggregation kinetics resulted in similar trends for all SWFs, with indices higher than for RWFs. The effect of milling on dough properties (i.e., mixing and leavening) was sample dependent. Overall, SWFs produced more gas, resulting in bread with higher specific volume. Bread crumb from SWF had higher lutein content in the wheat *cv* rich in xanthophylls, while bread from RWF of the blue-grained *cv* had a moderate but significantly higher content in esterified phenolic acids and total anthocyanins. In conclusion, there was no relevant advantage in using stone- as opposed to roller-milling (and vice versa).

**Keywords:** wholewheat flour; stone milling; roller milling; bioactive compounds; bread; pigmented wheat; dough rheology

#### **1. Introduction**

Whole grains have been (and still are in several countries) the most important energy source of mankind. They constitute a valuable and inexpensive source of numerous nutrients and phytochemicals, including fiber, phenolic compounds, minerals, and vitamins, mainly located in the germ and bran regions [1,2]. Although a universally accepted definition of whole grain has yet to be formulated [3], it is widely recognized that a whole grain product must contain bran, germ and endosperm in the same proportions as in the original, native grain [3]. Whatever the official definition is, the relationship between the consumption of whole grain foods and a lower incidence of diseases in the Occident has been suggested by numerous epidemiological studies [4–8].

Thanks to public institutions (governments and health organizations) and extensive promotional campaigns, consumers nowadays are fully aware of the numerous health benefits associated with the consumption of products made from whole grain flour (WF). Consequently, the demand for wholegrain products—in particular for staple foods such as bread and pasta—has been constantly growing [9]. This trend is recognizable even in countries where products made from refined flour have always been preferred for their undisputed superior sensory characteristics.

At present, two milling processes are mainly used to produce WF from wheat: (i) single–stream milling, and (ii) multiple-stream milling with recombination [10]. The former—where stone mills are used—is the world's oldest system for flour production. The stone mill is quite a simple machine, formed by two horizontal and overlapping grinding stones where the upper revolves while the other (bedstone) is stationary [11]. Grains are fed in the gap between the two stones and undergo shear, compression and abrasion forces. The ground fractions stay together during the entire milling process, and they are collected (with an extraction rate of 100%) at the bottom of the bedstone without separation according to particle size or composition [12].

Multiple-stream milling became established in the mid-1800s [11]. According to this process, wheat grains are progressively ground by means of a sequence of cast iron roller mills followed by sieving and sifting of the ground material [13]. Due to differences in composition, bran, germ and endosperm exhibit different friability and breakage patterns, thus facilitating the separation of bran and germ from flour, which derives from the endosperm. The separation of the three regions is enhanced by conditioning the kernels before milling [11,13]. The flour extraction rate is usually about 70% [12]. Even this milling process may lead to a WF if the different "fraction streams"—originating with the repeated grinding and sieving steps—are gathered at the end of the process. The extraction rate of the recombined WF should be about 100%, and the endosperm, germ and bran should be present in the same ratio as in the native whole grain [10]. Roller milling guarantees higher productivity, flexibility and more constant results over time; consequently, this process is highly preferred for industrial applications [13,14]. Moreover, the separation of bran and germ fractions during the process allows further treatments before recombination, such as mild heating and/or bran grinding, improving both technological performance and storage of the WF [13,15].

On the other hand, stone milling has been rediscovered in recent years by small farmers and bread-making artisans as it requires relatively low capital inputs [16]. Certainly, the simplicity and cheapness of the process (only one operation; grain tempering is not mandatory) also makes it suitable for household milling, favoring whole grain consumption for some segments of population [14]. Nevertheless, despite these advantages, stone milling not only is characterized by a lower yield but may also worsen the rheological properties of WF due to varying degrees of heat development [16,17] according to the type of small-scale mills used [14]. The negative effects of stone milling can be reduced by choosing suitable wheat *cultivars (cvs)* and/or farming procedures, as suggested by Gélinas et al. [18].

Despite the great interest in stone milling and increasing demands for WF-produced food, few studies have compared the effects of stone and roller milling on the same wheat varieties to identify and evaluate analogies and differences between the two kinds of WF [15,19]. Some research has found similarities in proximate chemical composition and phenolic profile of WF from stone milling (SWF) and roller milling (RWF) [17,20,21]. On the contrary, by promoting high heat development due to friction, compression and shear phenomena, stone milling leads to a significant loss in aminoacids and unsaturated fatty acids [17]. This worsening can affect not only the nutritional aspects of flour but also its technological properties, due to a partial denaturation of gluten proteins, relevant starch damage and/or differences in particle size distribution [15,17,20]. Specifically, SWFs exhibited lower pasting properties, higher water absorption, and lower stability compared to RWFs [14,21].

Regarding the relationship between milling process and bread quality, the data are not in agreement. Liu et al. [21] emphasized that RWFs exhibited the best steam-bread making performance, while according to Kihlberg et al. [22], bread produced using RWF was characterized by regular shape but higher compactness. Nevertheless, differences in bread volume in WFs obtained by the two types of milling can be resolved by adopting sourdough fermentation [14].

The few studies published so far on the effects of the milling process on WF and bread properties obtained by the same type of wheat presented no univocal findings and prompted our study to aim at giving a complete overview of behavior–from milling to baking–of WFs from four types of common wheat (*T. aestivum* L.), all belonging to the bread-making class and characterized by high protein content. Beyond these common traits, the four wheat samples differed in protein strength and physical structure of kernels–hard, semi-hard and soft endosperm–a property that highly impacts milling behavior and performance [23]. Moreover, two pigmented varieties were distinguished by their richness in bioactive compounds, differently located in the kernel (polyphenols and anthocyanins in the external layers and xanthophylls in the endosperm), whose retention has to be carefully ensured along the whole transformation chain.

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

#### *2.1. Wheat Samples*

The present study analyzed the SWF and RWF derived from four common wheat (*Triticum aestivum* L.) samples.

Table 1 compares the main information regarding the wheat samples. The two pigmented wheat *cvs* were chosen for their interesting bioactive compound content [24].

**Table 1.** Main information of common wheat samples.

TW, test weight; TKW, thousand kernel weight.

#### *2.2. Milling Procedures*