Meet the editors

Dr. Sinem Nalbantoglu is senior investigator in the field of molecular medicine and bioengineering, and chief senior researcher at the Laboratory of Molecular Oncology at the Gene Engineering and Biotechnology Institute, TUBITAK Marmara Research Center. She performed her postdoctoral studies at Georgetown University School of Medicine and National Institutes of Health. Dr. Nalbantoglu's research and teaching focus on systems and

molecular medicine, including omics-based clinical biomarkers for precision oncology and autoinflammation. She holds a TUBITAK R&D Women Entrepreneur Innovation Award, and works as principal investigator, senior researcher, and co-investigator of several partnering projects; she also supervises MSc and PhD students. She publishes her work in peer-reviewed national and international journals, and has authored/coauthored book chapters and conference proceedings. In addition, she has presented national/international invited talks, and has been a reviewer for many scientific journals.

Dr. Hakima Amri is a professor at Georgetown University School of Medicine in the Department of Biochemistry and Cellular and Molecular Biology and the Division of Integrative Physiology. She holds a Master of Science degree in Reproductive Physiology and a PhD in Steroid Biochemistry from Pierre and Marie Curie University-Sorbonne, Paris, France. She then moved to the United States, where she joined the Georgetown University Faculty.

Dr. Amri's research focus is on understanding the action mechanism of disease processes using comprehensive systems biology approaches. She investigates cancer biomarkers and bio-signature identification using big data. Her project is enhanced by the novel analytical approach she recently developed in collaboration with her colleagues. She uses this novel method to translate cancer omics, from genomics, proteomics, and metabolomics high-throughput data, into molecular signatures that are then presented in a multidimensional and dynamic model best suited for precision medicine. Dr. Amri is currently working on translating Phylomics® (http://phylomics.com) into a cost-effective and noninvasive diagnostic tool. Her work is supported by a recently granted patent by the US Patents Office. Dr. Amri has taught a significant number of students in classroom and laboratory settings, spanning undergraduate, graduate, doctoral, and postdoctoral levels. She received funding from both government and private sectors. Her scholarly activities are marked by her published work in peer-reviewed journals, presentations at national and international conferences, and authorship of a book on Avicenna's medicine, several book chapters, and reviews.

Contents

Medicine

Alzheimer's Disease

*by Munindra Ruwali*

Hyperpigmentation

*Pingshen Hu and Xiaodong Su*

*and Gustavo Acosta-Altamirano*

*by Sinem Nalbantoglu*

Cancer

**Preface III**

**Chapter 1 1**

**Chapter 2 11**

**Chapter 3 35**

**Chapter 4 49**

**Chapter 5 71**

**Chapter 6 89** A Novel P53/POMC/Gas/SASH1 Autoregulatory Feedback Loop and Pathologic

**Chapter 7 113**

**Chapter 8 137**

Energy Metabolism Heterogeneity-Based Molecular Biomarkers for Ovarian

*by Ding'an Zhou, Jiawei Zeng, Xing Zeng, Yadong Li, Zhixiong Wu, Xin Wan,*

Introductory Chapter: Insight into the OMICS Technologies and Molecular

Precision Medicine: Role of Biomarkers in Early Prediction and Diagnosis of

*by Liming Shen, Sijian Xia, Huajie Zhang, Fang Yao, Xukun Liu, Yuxi Zhao,*

Pharmacogenetics and Cancer Treatment: Progress and Prospects

*by Sinem Nalbantoglu and Abdullah Karadag*

CTCs as Liquid Biopsy: Where Are We Now? *by Laure Cayrefourcq and Catherine Alix-Panabières*

*by Na Li, Xiaohan Zhan and Xianquan Zhan*

Molecular Diagnosis of Invasive Aspergillosis

Metabolomics: Basic Principles and Strategies

*by María del Rocío Reyes-Montes, Esperanza Duarte-Escalante, María Guadalupe Frías-De-León, Erick Obed Martínez-Herrera*

*Ming Ying, Javed Iqbal and Qiong Liu*

## Contents

*by Sinem Nalbantoglu*

Preface

Molecular medicine is an applied science focused on human genes/transcripts, proteins, metabolites, and metabolic networks that describe molecular and cellular processes of health and disease onset and progression. Molecular medicine aims to define associated predictive, diagnostic, and treatment assessment biomarkers for personalized diagnostic/therapeutic strategies, and for a targeted approach to providing the best healthcare for the twenty-first century. Here, in *Molecular Medicine*, a collection of scientific contributions addressing specifically the field of molecular biology and genetics, genetic epidemiology, laboratory medicine, molecular diagnosis, next-generation sequencing, mass spectrometry, omics technologies, biomarker discovery, liquid biopsy, cell heterogeneity, circulating tumor cells, cancer, *inflammation,* infectious diseases, systemic and chronic diseases, neurodegenerative disorders, pharmacogenetics and pharmacogenomics, and precision/personalized

In the first chapter entitled "Introductory Chapter: Insight into the OMICS Technologies and Molecular Medicine" by Sinem Nalbantoglu and Abdullah Karadag, an overview of the latest headlines of molecular medicine, including promising research strategies and their emerging roles in biomedical research, has been compiled. Throughout the chapter, primary objectives of molecular medicine, involving prediction of potential future pathologies, identifying disease state through effective screening and early diagnosis systems, decision-making on effective treatment strategies, monitoring prognosis and health status, as well as predicting recurrence earlier to apply alternative treatments, have been

In the chapter "Precision Medicine—Role of Biomarkers in Early Prediction and Diagnosis of Alzheimer's Disease" by Liming Shen, Sijian Xia, Huajie Zhang, Fang Yao, Xukun Liu, Yuxi Zhao, Ming Ying, Javed Iqbal, and Qiong Liu, the authors precisely outline clinical biomarkers and precision medicine for Alzheimer's disease, which is a chronic and irreversible neurodegenerative disorder. In this chapter, the authors introduce promising research efforts and progress in the development and validation of invasive, minimally invasive, and non-invasive personalized diagnostic strategies and their applications, neuroimaging techniques and neurochemical assays, novel ultrasensitive immunoassay, genetic testing, mass spectrometry methods, metabolomics, and

Munindra Ruwali, the author of the chapter "Pharmacogenetics and Cancer Treatment: Progress and Prospects," contributes a comprehensive review on the developments and recent advances in cancer research, pharmacogenetics, and oncogenetics. As precisely addressed by the author throughout the chapter, cancer pharmacogenetics has great importance for considering genotypic and phenotypic heterogeneity among patients, and offering personalized treatment leads to improved response to therapy and decreased side effects in

medicine is presented.

emphasized.

exosomes.

individuals.


**Abstract**

The order Diptera includes all true flies. Members of this order are the most ecologically diverse and probably have a greater economic impact on humans than any other group of insects. The application of explicit methods of phylogenetic and morphological analysis has revealed weaknesses in the traditional classification of dipteran insects, but little progress has been made to achieve a robust, stable classification that reflects evolutionary relationships and morphological adaptations for a more precise understanding of their developmental biology and behavioral ecology. The current status of Diptera phylogenetics is reviewed in this chapter. Also, key aspects of the morphology of the different life stages of the flies, particularly characters useful for taxonomic purposes and for an understanding of the group's biology have been described with an emphasis on newer contributions and progress in understanding this important group of insects.

**Keywords:** Tephritoidea, Diptera flies, Nematocera, Brachycera metamorphosis, larva


3.4. Aflatoxins

content was achieved when B. subtilis RC 218 and Brevibacillus spp. RC 263 were applied at anthesis for two seasons [144] which was consistent with previous findings under greenhouse conditions by the same authors [163], although there was no constant reduction in the disease incidence. Opposite to that, Khan and Doohan tested three strains of Pseudomonas spp., two strains of fluorescens and one strain of frederiksbergensis, against F. culmorum and DON production in wheat and barley in a small scale field experiment. The results showed that DON was

Other types of trichothecenes were not well researched as the previously mentioned toxins due to their low incidence in crops. Variable results for T-2 toxin after spraying the ears of susceptible and resistant wheat cultivars with Trichoderma spp. under greenhouse conditions were documented. The author used four fungi, Epicoccum spp., Trichoderma spp., Penicillium spp. and Alternaria spp. however the last one is known for production of Alternaria toxins [107].

Although zearalenone (ZEN) is an important mycotoxin in many cereals, less attention has been paid to control this toxin. ZEN is a potent mycoestrogen which competitively binds to estrogen receptors causing reproductive disorders in farm animals and human [5]. Other forms of ZEN include α and β zearalenol, zearalanone and, α and β –zearalanol which are

Trichoderma isolates have recently been reported to detoxify ZEN by transforming ZEN into reduced and sulfated forms [165]. This was in accordance with previous results by Gromadzka et al. who tested two isolates of Trichoderma and several isolates of Clonostachys in vitro against two isolates of F. graminearum and two isolates of F. culmorum. Despite the high rate of ZEN reduction (over 96%), the performance of these isolates under greenhouse or field experiments

C. rosea converts ZEN into less toxic compounds through an enzymatic alkaline hydrolysis by lactonohydrolase in vitro [23, 166]. This has been proved after cloning the coding region of the responsible gene, zhd 101, and expressing in Schizosaccharomyces pombe [167] and Escherichia coli, but not with Saccharomyces cerevisiae which exhibited weak detoxification activity against ZEN [168]. Through this approach which involves the direct interaction between BCAs and pathogen toxin, resistance of BCAs to mycotoxin itself is an important feature to ensure the efficacy and durability. Also, it was proven that C. rosea is tolerant to ZEN exposure due to the

Fumonisin B1 (FB1), the main member of fumonisins, is produced by F. verticillioides and F. proliferatum which usually infect maize [14]. The mycotoxin suppresses ceramide synthase and causes neurological toxicities in horses, pulmonary edema in pigs, and may pose hepatotoxicity and esophageal cancer in human [18]. Therefore, several trials have been conducted to effectively control the mycotoxin in maize using different strategies. Most of the field studies were done using bacterial BCAs [147, 148, 150, 158] while other types of BCAs, and fungi, were

reduced in wheat and barley by 12 and 21%, respectively [164].

often detected at variable concentration usually lower than ZEN.

presence of high numbers of ATP-binding cassette transporters [169].

3.2. Zearalenone

70 Mycotoxins - Impact and Management Strategies

was not confirmed [128].

3.3. Fumonisins

AFs are the most natural carcinogenic substance in the history targeting mainly liver and are classified as Group 1 according to the International Agency for Research on Cancer [4, 6, 16, 171]. A. flavus and A. parasiticus infect mostly groundnuts, maize, cottonseed, soybean and tree nuts in the field and/or during storage producing a wide range of secondary toxic metabolites including AFs [60, 172]. Researchers have mostly been focusing on A. flavus as the fungus is highly invasive and more widespread in nature compared to A. parasiticus. Regarding their ability to synthetize mycotoxins, toxigenic A. flavus strains produce aflatoxin B1 (AFB1) and B2 (AFB2) while A. parasiticus produces four types of AFs (AFB1, AFB2, AFG1 and AFG2). CPA is only produced by A. flavus including strains which lack the potential to produce AFs [173].

In general, reduction of AFs in different crops has mostly been performed with nontoxigenic A. flavus strains [27, 52, 54, 60, 65, 114, 120, 123]. Some of these strains (AF36 as an example) are commercially available in the market [53, 65]. Two theories are suggested on the mode of action for the reduction of AFs by non-toxigenic A. flavus BCAs; (i) reduction due to competitive exclusion on toxigenic wild A. flavus population and (ii) inhibition of biosynthetic pathways involved in aflatoxin production, however the exact mechanism is still obscure [62].

Doster et al. used A. flavus strain AF36 as a BCA to control AFs in pistachio orchards for four consecutive seasons (2008–2011) and he could diminish AFs level by 20–45% [114]. In groundnuts, more trials in vitro [61, 66] and in the field [58–60] have been done. Zhou et al. 2015 found a positive correlation between AFs reduction rate and inoculum dose while Hulikunte Mallikarjunaiah et al. 2017 measured total AFs in rhizospheric and geocarpospheric soil and groundnut seeds after he treated them with two strains isolated from India. A significant reduction of mycotoxin concentration below the maximum permissible levels for ground nuts was obtained [61]. Field trials in Argentina were designed to control AFs in groundnut. However, the author reported a high level of AFs reduction, and the results were inconsistent between the two seasons [58, 59].

delivery (conidial or spore suspension/with or without carrier), application time (during seeding or flowering) and application route (to the soil or directly to the seed) to ensure the interaction of BCAs against the pathogen. Examples for the available BCAs in the market include AF36 and Afla-Guard® which are commercial BCAs for pre-harvest application to control aflatoxin contamination in the United States [62], Polyversum®, a recent authorized commercial product in France (Pythium oligandrum strain ATCC 38472) to be used against Alternaria spp., Fusarium spp., and other plant pathogens, and Plant ShieldTM which is the

Biological Control of Mycotoxigenic Fungi and Their Toxins: An Update for the Pre-Harvest Approach

http://dx.doi.org/10.5772/intechopen.76342

73

It is crucial to test all the application related parameters in the field as these parameters may give significantly variable results which are not usually followed in many of the performed field trials against mycotoxigenic caused diseases. For example, point inoculation of Streptomyces sp. BN1 was not effective to control FHB in wheat while spraying of bacterial spores during wheat flowering gives better results [175]. Successful formulation of C. rosea ACM941 guaranteed its efficacy to control FHB in corn, soybean and wheat under filed conditions [176], while most of the field trials used a conidial or spore suspension of the BCAs which may give variable and inconsistent results. Ear inoculation with B. amyloliquefaciens and Enterobacter hormaechei exhibit highly changeable results while treatment of seeds showed more stable results for managing F. verticillioides infection and toxin content in maize [150]. On the other hand, B. subtilis strains SB01, SB04, SB23, and SB24 were performing better to control root rot disease when they were applied to soil than treatment of soybean seeds [145]. Omitting one or

In some cases, a mixture of two more BCAs maybe advisable in the field for a better disease control in case they have a synergistic effect. For example, mixture of L. plantarum SLG17 and B. amyloliquefaciens FLN13 showed more efficacy in controlling FHB in wheat durum [131].

Although the field trials are exhausting and time consuming, it should consider the application way, application time, effective dose and the best formula in order to precisely evaluate the performance of the selected BCAs and thereafter ensure an effective control of the mycotoxi-

An important obstacle in the commercialization of BCAs is legislation. Current legislations in Europe classify BCAs as Plant Protection Products/Pesticides and hence they must follow the according regulations of the pesticides. This entails that for each BCA the mode of action must

Despite the considerable amount of research that have been done to screen and select effective BCAs to control mycotoxigenic pathogens and their mycotoxins, still there are several pitfalls for using BCAs. For instance, the broad spectrum antagonistic activity of some BCAs such as Trichoderma spp., against several pathogenic fungi may also affect other beneficial organisms present in rhizosphere [178] and this may require more research for target specific BCAs. Even though implementation of a biological control strategy is strongly recommended to replace the

more of the above parameters may lead to misevaluation of the selected BCAs.

registered product for T. harzianum 22.

genic fungal infection and their mycotoxins.

be documented and their use should be rational [177].

5. Conclusions and future perspectives

High levels of AFs and CPA control in maize field were achieved after challenging two strains of A. flavus with atoxigenic strains K49 and NRRL 21882 [65]. Mauro et al. could obtain similar results in vitro after screening for local atoxigenic strains from Italy [67]. In Nigeria, a successful maize field trial exhibited the promising use of two locally isolated strains, La3279 and La3303, in controlling AFB1 and AFB2 up to 99.9% [120]. When these two strains mixed with other two strains to make a mixture applied to the soil before flowering, a similar conclusion was obtained [55] with the advantage of persistence of the biocontrol effect during storage.

Researchers have also tested different species of Trichoderma such as T. viride, T. harzianum and T. asperellum [38, 95, 115, 116]; bacteria [84, 121, 124]; yeast [36, 174]; and algae [118] as a potential alternative BCAs to control Aspergillus spp., although not all have looked into mycotoxins (Figure 2B). Production of two volatile compounds, dimethyl trisulfide and 2,4-bis(1,1 dimethylethyl)-phenol, by Shewanella algae strain YM8 showed a 100% inhibition on aflatoxin synthesis in maize and peanuts stored at different water activities [118]. Previously, B. subtilis RCB 90 in vitro was also reported to completely inhibit AFB1 [121]. The yeast, Candida parapsilosis IP1698 was also able to inhibit aflatoxin production (90–99%) at different pH and temperatures [174]. This was also in line with the same reduction percentage obtained but with Bacillus spp. P1 and Bacillus spp. P11 [40]. Aiyaz et al. tested in the field, four BCAs and all the formulations, by maize seeds treatment application, had a significant reduction in AFs level [95].


*Article* **Feasibility of Defatted Juice from Sea-Buckthorn Berries (***Hippophae rhamnoides* **L.) as a Wheat Beer Enhancer**

**Justyna Belcar \* and Józef Gorzelany**

Department of Food and Agriculture Production Engineering, University of Rzeszow, 4 Zelwerowicza Street, 35-601 Rzeszów, Poland<EMAIL_ADDRESS>
**\*** Correspondence<EMAIL_ADDRESS>
**Abstract:** Juice made from sea-buckthorn berries (*Hippophae rhamnoides* L.) is a valuable source of bioactive compounds, vitamins, as well as micro- and macronutrients. By applying defatted seabuckthorn juice, it is possible to enhance wheat beer and change its sensory properties and the contents of bioactive compounds in the finished product. A sensory assessment showed that wheat beers with a 5% *v*/*v* addition of sea-buckthorn juice were characterised by a balanced taste and aroma (overall impression). Physicochemical analyses showed that, compared to the control samples, wheat beers enhanced with defatted sea-buckthorn juice at a rate of 5% *v*/*v* or 10% *v*/*v* had high total acidity with respective mean values of 5.30 and 6.88 (0.1 M NaOH/100 mL), energy values lower on average by 4.04% and 8.35%, respective polyphenol contents of 274.1 mg GAE/L and 249.7 mg GAE/L, as well as higher antioxidant activity (measured using DPPH, FRAP, and ABTS assays). The findings show that the samples of wheat beer enhanced with sea-buckthorn juice had average ascorbic acid contents of 2.5 and 4.5 mg/100 mL (in samples with 5% *v*/*v* and 10% *v*/*v* additions, respectively) and contained flavone glycosides, e.g., kaempferol-3-O-glucuronide-7-O-hexoside. Based on the current findings, it can be concluded that wheat beer enhanced with sea-buckthorn juice could emerge as a new trend in the brewing industry.

**Keywords:** sea-buckthorn; defatted juice; wheat beer; beer quality; bioactive compounds; antioxidant potential of beer

#### **1. Introduction**

Beer is a type of beverage which contains four main ingredients: malt, hops, water, and yeast. In wheat beers, some of the barley malt (most commonly from 40 to 60% of the total input material) is replaced with wheat malt or unmalted wheat grain [1]. Wheat beers are characterised by an original flavour owing to the wide range of chemical compounds (e.g., phenols, aldehydes, and esters and their derivatives) produced in the process of top fermentation that contribute to the flavour, which can resemble vanilla, cloves, bananas, or fresh fruit; the effect is produced by the interaction of the two types of malt (barley and wheat) as well as the addition of hops. The final product of the brewing process is characterised by delicate and stable frothy foam, a slightly bitter taste, and haziness [2–4]. Wheat beers also have high contents of antioxidant compounds including polyphenols [5].

In recent years, a trend has been observed in consumers' increasing preference for fruit beers, which mainly include morello cherry, raspberry, banana, and strawberry, as well as other exotic fruit beers. The fruit component may be introduced by adding pulp, juice, concentrate, or aroma, most commonly during the fermentation process. By enhancing beer products with fruit, it is possible to improve their sensory qualities (such as colour, aroma, and taste), and to increase the health-promoting properties of the beverage, which are linked to higher contents of bioactive compounds (e.g., polyphenols), resulting in the higher antioxidant activity of fruit beers compared to traditional beer products [6,7]. In global markets we can encounter the very popular Radler-style beverages, i.e., a combination

**Citation:** Belcar, J.; Gorzelany, J. Feasibility of Defatted Juice from Sea-Buckthorn Berries (*Hippophae rhamnoides* L.) as a Wheat Beer Enhancer. *Molecules* **2022**, *27*, 3916. https://doi.org/10.3390/ molecules27123916

Academic Editor: Francesco Cacciola

Received: 30 May 2022 Accepted: 16 June 2022 Published: 18 June 2022

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

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

of beer and flavoured sugar syrup or fruit juice [8,9], as well as Belgian specialty beers produced as a result of spontaneous fermentation with the addition of raspberries or morello cherries, respectively, known as 'Framboise' and 'Kriek' Lambic beer [6,10,11].

The fruit of sea-buckthorn (*Hippophae rhamnoides* L.), depending on the variety, are yellow to orange in colour, which results from the high concentrations of carotenoids (including lutein, carotene, and zeaxanthin). Sea-buckthorn berries have also been reported to have high contents of health-promoting compounds such as polyphenols (e.g., flavanols and chlorogenic acid), organic acids, micro- and macronutrients, and vitamins [12–15], including very high levels of ascorbic acid (on average from 53 up to 1550 mg·100 g−1 [14,16]), and they do not contain ascorbinase enzyme responsible for the decomposition of ascorbic acid [17]. Owing to their contents of terpenes, alcohols, tannins, and aldehydes, seabuckthorn berries have a characteristic aroma [12].

The extraction of juice from sea-buckthorn berries is a complex process that may produce changes in the chemical composition and bioavailability of nutrients in the final product. Depending on the preservation process applied (e.g., high-temperature short-term method; HTST), the changes that take place affect the sensory properties, mainly the taste of the juice; on the other hand, a short-term thermal treatment process does not lead to the degradation of ascorbic acid, the content of which decreases during the production of the juice by about 5–11% in relation to the amount of this compound in fresh fruit. Additional technological processes, such as filtration and clarification, contribute to a decrease in the ascorbic acid content. The high-pressure processing (200–600 MPa) that is applied to preserve juice does not produce changes in the quality of the final product; however, it results in a reduced size of particles contained in the juice, enhancing the yellow-orange colour of sea-buckthorn juice [18,19]. Both sea-buckthorn berries and seabuckthorn juice contain fatty acids, including oleic and palmitoleic acids, phospholipids, and phytosterols [17]. Because these compounds are present, sea-buckthorn juice must be separated into an aqueous fraction and an oil fraction. Lipids present in the oil fraction bind into protein–lipid complexes with soluble low-molecular-weight proteins originating from the malts and they reduce the stability of the structure of the beer head, which comprises carbon dioxide molecules [3,4,20]. The high total acidity of sea-buckthorn juice, which is on average in the range of 2.1–9.1 g·100 mL−1 depending on the variety of the raw material, is significantly related to the content of malic and quinic acid (90% share in organic acids; [19]). The bitter and pungent taste of sea-buckthorn juice can be balanced in combination with other beverages, including tea, coffee, wine, and beer [21]. The addition of defatted seabuckthorn juice to wheat beer, which is characterised by a delicate, slightly sweet taste, may be an interesting and original option acceptable for consumers.

The purpose of this study was to identify the physicochemical, sensory, and antioxidant properties of wheat beers produced with the addition of defatted sea-buckthorn juice. The study also assessed the applicability of the findings to expand the assortment of fruit beers and to make use of sea-buckthorn berries in a new sector of the food industry.

#### **2. Results and Discussion**

#### *2.1. Physicochemical Characteristics of the Wheat Beers*

The findings describing the physicochemical parameters of wheat beers enhanced with defatted sea-buckthorn juice are shown in Table 1.

The contents of the apparent extract in the wheat beer samples were in the range of 3.33–4.06% m/m; significantly higher contents of the apparent extract were found in the beer samples with defatted sea-buckthorn juice added at a rate of 10% *v*/*v* (E10 and L10, Table 1). The highest contents of real extract and original extract were identified in the control samples (E0 and L0; Table 1).

**Table 1.** Results of physicochemical analysis of wheat beers with defatted sea-buckthorn juice added.

Data are expressed as mean value (*n* = 3) ± SD; SD—standard deviation. Mean values within a row with different letters are significantly different (*p* < 0.05). E—'Elixer' cultivar; L—'Lawina' cultivar; 0—wheat beer without defatted sea buckthorn juice; 5—wheat beer with 5% *v*/*v* defatted sea buckthorn juice; 10—wheat beer with 10% *v*/*v* defatted sea buckthorn juice.

The course of the fermentation process and the degree of the final fermentation affect the content of ethyl alcohol, the basic component of beer-type beverages, which is responsible for the sensory characteristics of beer that are perceived by consumers [22]. The degree of the final apparent fermentation in the wheat beer samples ranged from 69.88 to 77.62%, with the highest values identified in the control samples (E0 and L0). An increase in the concentration of defatted sea-buckthorn juice led to a significant decrease in the final apparent fermentation by an average of 3.69% in the samples with a 5% *v*/*v* addition of sea-buckthorn juice and by 8.33% in the samples with sea-buckthorn juice added at a rate of 10% *v*/*v* (Table 1). The values of the final true fermentation identified in the wheat beer samples were less varied but statistically different, independent from the beer samples acquired from wheat malt produced from grains of 'Lawina' and 'Elixer' wheat varieties. Similar to the degree of the final fermentation, the highest alcohol contents were identified in the control samples (E0 and L0), whereas the beer samples with defatted seabuckthorn juice added at a rate of 5% *v*/*v* and 10% *v*/*v* were found with ethanol contents that were lower on average by 4.02% and by 9.19%, respectively (Table 1). According to Gasi ´nski et al. [23], fruit beer should have a higher ethyl alcohol content compared to beer that is not enhanced with fruit. The lower contents of ethyl alcohol in the investigated wheat beer samples could be linked to the addition of defatted sea-buckthorn juice, which contains relatively low concentrations of total sugars (on average 4.94–5.72% relative to the variety) and reducing sugars (on average 1.59–1.83% relative to the variety; [13]); these are processed by the yeast in the fermentation process only to a small degree. Furthermore, the addition of sea-buckthorn juice led to an increase in the volume of the finished beer product while decreasing the concentration of the ethanol in the investigated wheat beer samples. A study by Nordini and Garaguso [10] showed that apple beer had an alcohol content of 5.2% *v*/*v*, whereas beer samples enriched with orange peel were found with an ethanol content of 6.0% *v*/*v*. On the other hand, Baigts-Allende et al. [6] reported an alcohol content of 4.0–8.2% *v*/*v* in citrus beer and 2.5–3.5% *v*/*v* in apple beer. Yang et al. [7] investigated apple beer and cranberry beer and reported ethanol contents of 3.5% *v*/*v* and 3.6% *v*/*v*, respectively. Patra¸scu et al. [9] reported ethanol contents in lemon beer samples in the range of 1.9–4.0% *v*/*v*, in grapefruit beer samples of 1.9–2.5% *v*/*v*, and in cranberry beer samples of 4.0% *v*/*v*. The high ethanol content of the investigated wheat beers, in particular in the control samples (E0 and L0), corresponded to a relatively high calorific value of the finished product, which ranged from 54.48 to 57.22 kcal/100 mL; on the other hand, the addition of defatted sea-buckthorn juice led to a decrease in the calorific value of the wheat beer samples by an average of 6.2% (Table 1).

Wheat beer as a rule is darker in colour compared to barley beer (depending on the beer style). The wheat beer samples produced without the addition of defatted seabuckthorn juice (E0 and L0) were found with a slightly darker colour; however, an increased concentration of sea-buckthorn juice added to the beer led to a lighter colour in the wheat beer samples (Figures 1 and 2; Table 1). Baigts-Allende et al. [6] reported that barley beer produced with the addition of citric fruit was found with a colour of 5.8 EBC units, whereas apple beers were characterised by a slightly stronger colour in the range of 6.34–9.81 EBC units. Patra¸scu et al. [9] assessed the colour of lemon, grapefruit, and cranberry beers and reported respective values of 6.75–6.83 EBC units, 16.98–17.36 EBC units, and 5.55 EBC units. beer samples of 4.0% *v*/*v*. The high ethanol content of the investigated wheat beers, in particular in the control samples (E0 and L0), corresponded to a relatively high calorific value of the finished product, which ranged from 54.48 to 57.22 kcal/100 mL; on the other hand, the addition of defatted sea‐ buckthorn juice led to a decrease in the calorific value of the wheat beer samples by an average of 6.2% (Table 1). Wheat beer as a rule is darker in colour compared to barley beer (depending on the beer style). The wheat beer samples produced without the addition of defatted sea‐buckthorn juice (E0 and L0) were found with a slightly darker colour; however, an increased concentration of sea‐buckthorn juice added to the beer led to a lighter colour in the wheat beer samples (Figures 1 and 2; Table 1). Baigts‐Allende et al. [6] reported that barley beer produced with the addition of citric fruit was found with a colour of 5.8 EBC units, whereas apple beers were characterised by a slightly stronger colour in the range of 6.34–9.81 EBC units. Patraşcu et al. [9] assessed the colour of lemon, grapefruit, and cranberry beers and reported respective values of 6.75–6.83 EBC units, 16.98–17.36 EBC units, and 5.55 EBC units.

*Molecules* **2022**, *27*, x FOR PEER REVIEW 4 of 20

rate of 5% *v*/*v* and 10% *v*/*v* were found with ethanol contents that were lower on average by 4.02% and by 9.19%, respectively (Table 1). According to Gasiński et al. [23], fruit beer should have a higher ethyl alcohol content compared to beer that is not enhanced with fruit. The lower contents of ethyl alcohol in the investigated wheat beer samples could be linked to the addition of defatted sea‐buckthorn juice, which contains relatively low concen‐ trations of total sugars (on average 4.94–5.72% relative to the variety) and reducing sugars (on average 1.59–1.83% relative to the variety; [13]); these are processed by the yeast in the fermentation process only to a small degree. Furthermore, the addition of sea‐buckthorn juice led to an increase in the volume of the finished beer product while decreasing the concentration of the ethanol in the investigated wheat beer samples. A study by Nordini and Garaguso [10] showed that apple beer had an alcohol content of 5.2% *v*/*v*, whereas beer samples enriched with orange peel were found with an ethanol content of 6.0% *v*/*v*. On the other hand, Baigts‐Allende et al. [6] reported an alcohol content of 4.0–8.2% *v*/*v* in citrus beer and 2.5–3.5% *v*/*v* in apple beer. Yang et al. [7] investigated apple beer and cranberry beer and reported ethanol contents of 3.5% *v*/*v* and 3.6% *v*/*v*, respectively. Patraşcu et al. [9] reported ethanol contents in lemon beer samples in the range of 1.9–4.0% *v*/*v*, in grapefruit beer samples of 1.9–2.5% *v*/*v*, and in cranberry

The addition of defatted sea‐buckthorn juice to wheat beer led to a decrease in the pH value by an average of 13.51% in the samples with a 5% *v*/*v* addition of sea‐buckthorn juice and by an average of 18.74% in the samples with a 10% *v*/*v* addition of sea‐buckthorn juice relative to the control samples (E0 and L0), which corresponded to a significant increase in the acidity (even twofold in the case of the samples with sea‐buckthorn juice added at a rate of 10% *v*/*v*; Table 1). Adadi et al. [24] reported that the pH value and acidity of beer enriched with sea‐buckthorn berries

samples enhanced with orange peel were found with a pH value of 4.86. Patraşcu et al. [9] investigated lemon, grapefruit, and cranberry beers, which were found with an acidity of 4.0–4.64; 4.0–4.4; and 3.76, respectively, whereas the pH values in these types of beer were found to be 2.85– 3.09, 3.27–3.49, and 2.91, respectively. A lower pH in beer results in a reduced growth of undesirable microflora, and consequently leads to the greater microbiological stability of the finished beer product [23]. The addition of sea‐buckthorn juice, characterised by high acidity and a low pH, on the seventh day during the fermentation process further reduces the risk of microbiological contamination, which is of great importance in the

All the wheat beer samples were found with similar contents of carbon dioxide (0.44–0.48%; Table 1). Patraşcu et al. [9] reported contents of carbon dioxide in lemon beer samples in the range of 0.48–0.55%, in grapefruit beer samples of 0.52%, and in cranberry beer samples of 0.55%. The contents of bitter substances in the wheat beer samples enhanced with defatted sea‐buckthorn juice were at a similar level (17.5–19.7 IBU;

**Figure 2.** The appearance of the obtained wheat beers (from left)—control (L0); with 5% *v*/*v* addition of defatted sea‐buckthorn juice (L5); and with 10% *v*/*v* addition of defatted sea‐buckthorn juice (L10). **Figure 2.** The appearance of the obtained wheat beers (from left)—control (L0); with 5% *v*/*v* addition of defatted sea-buckthorn juice (L5); and with 10% *v*/*v* addition of defatted sea-buckthorn juice (L10).

production of fruit beer.

The addition of defatted sea-buckthorn juice to wheat beer led to a decrease in the pH value by an average of 13.51% in the samples with a 5% *v*/*v* addition of sea-buckthorn juice and by an average of 18.74% in the samples with a 10% *v*/*v* addition of sea-buckthorn juice relative to the control samples (E0 and L0), which corresponded to a significant increase in the acidity (even twofold in the case of the samples with sea-buckthorn juice added at a rate of 10% *v*/*v*; Table 1). Adadi et al. [24] reported that the pH value and acidity of beer enriched with sea-buckthorn berries amounted to 3.9 and 2.2, respectively. A study by Nordini and Garaguso [10] reported that apple beer samples had a pH of 4.42, whereas beer samples enhanced with orange peel were found with a pH value of 4.86. Patra¸scu et al. [9] investigated lemon, grapefruit, and cranberry beers, which were found with an acidity of 4.0–4.64; 4.0–4.4; and 3.76, respectively, whereas the pH values in these types of beer were found to be 2.85–3.09, 3.27–3.49, and 2.91, respectively. A lower pH in beer results in a reduced growth of undesirable microflora, and consequently leads to the greater microbiological stability of the finished beer product [23]. The addition of sea-buckthorn juice, characterised by high acidity and a low pH, on the seventh day during the fermentation process further reduces the risk of microbiological contamination, which is of great importance in the production of fruit beer.

All the wheat beer samples were found with similar contents of carbon dioxide (0.44–0.48%; Table 1). Patra¸scu et al. [9] reported contents of carbon dioxide in lemon beer samples in the range of 0.48–0.55%, in grapefruit beer samples of 0.52%, and in cranberry beer samples of 0.55%. The contents of bitter substances in the wheat beer samples enhanced with defatted sea-buckthorn juice were at a similar level (17.5–19.7 IBU; Table 1) and the value increased with a higher addition of sea-buckthorn juice and was significantly greater compared to the wheat beer control samples (E0 and L0; Table 1). The bitter taste in the investigated beer samples originates not only from the basic raw material used in the production of the beer (i.e., hops) but also from the added sea-buckthorn juice (polyphenolic compounds present in juice). The bitter taste and contents of bitter substances in beer are significantly impacted by the variety and dose of the hops applied, the degree of isomerisation of α-acids during the process of boiling the wort with hops, and the reaction of proteins with polyphenols contained in the malt [2,25]. Sea-buckthorn berries contain terpenoids, tannins, as well as aldehydes and alcohols, which are responsible for the bitter taste and characteristic aroma in products enhanced with sea-buckthorn berries [12].

#### *2.2. Contents of Bioactive Compounds in Wheat Beers*

Ascorbic acid is a chemical compound known for its antioxidant properties. The effects produced by ascorbic acid include the strengthening of defence mechanisms; it is also involved in the synthesis of collagen and the absorption of iron in the human body and it promotes the transcription of mRNA and the treatment of scurvy [18,26]. Sea-buckthorn berries were reported to have high contents of ascorbic acid, ranging from 53–131 mg·100−1 g [14] to 114–1550 mg·100 g−1 [16], depending on the sea-buckthorn variety and the timing of the harvest; on the other hand, they do not contain the ascorbinase enzyme involved in the decomposition of ascorbic acid in fruit and vegetables. Wheat beer samples enhanced with defatted sea-buckthorn juice had low contents of ascorbic acid, and this compound was not identified in the E0 and L0 beer samples (Table 2). Because of the very rapid decomposition of ascorbic acid, this compound is generally not found in fruit beers (e.g., those with the addition of lemon, grapefruit, black currant, and strawberry), although the fruit added to the beer typically has high contents of ascorbic acid (the respective reported values being 25–53 mg·100 g−1 ; 4–34.4 mg·100 g−1 ; 181–215 mg·100 g−1 ; and 41.2–60 mg·100 g−1 [27]). A study by Pimentel et al. [28] used camu-camu fruit (ascorbic acid contents in the range of 2.4–3 g·100 g−1 of fruit) to enhance Witbier-type beer. The beer was found with an ascorbic acid content of 15.8 mg·100 mL−1 .

The contents of polyphenols, bitter substances, vitamins, and melanoidins impact the antioxidant potential of beer [29,30]. The addition of sea-buckthorn juice to wheat beer significantly increased the antioxidant activity of the beer samples assessed using

three methods (DPPH·, FRAP, ABTS+.), and the increase was associated with a higher concentration of defatted sea-buckthorn juice in the wheat beer samples (Table 2). In comparison, Nordini and Garaguso [10] reported that beer samples produced with the addition of orange peel were found with an antioxidant capacity, assessed using ABTS, of 2.67 mM TE/L, and a reducing capacity, shown by a FRAP assay, of 5.65 mM Fe2+/L. They also applied FRAP and ABTS assays to measure the antioxidant capacity of apple beer and reported the respective values of 3.08 mM Fe2+/L and 1.62 mM TE/L. The addition of juice from persimmons (kaki) to barley beer led to a decrease in the antioxidant capacity, measured using an ABTS assay, from 6.36 mM TE/L (in a control sample made from 100% barley wort) to 1.65 mM TE/L (in samples made from 25% wort and 75% kaki fruit juice [11]). Deng et al. [31] enhanced beer with omija fruit added during the fermentation process and reported antioxidant activity, measured by DPPH assay, of 1.68 mM TE/L, and a reducing capacity, assessed with FRAP, of 2.4 mM Fe2+/L. Portuguese commercial fruit beers flavoured with lemon were reported to have antioxidant capacity in the range of 0.035–0.037 mM TE/L, according to DPPH assay, and a level of 0.008 mM TE/L, according to an ABTS assay [32].

**Table 2.** Content of ascorbic acid and antioxidant activity of wheat beers.

Data are expressed as mean value (*n* = 3) ± SD; SD—standard deviation. Mean values within a row with different letters are significantly different (*p* < 0.05). E—'Elixer' cultivar; L—'Lawina' cultivar; 0—wheat beer without defatted sea buckthorn juice; 5—wheat beer with 5% *v*/*v* defatted sea buckthorn juice; 10—wheat beer with 10% *v*/*v* defatted sea buckthorn juice; n.d.—not detected; TE—expressed as Trolox equivalent (mM TE/L).

Polyphenolic compounds occurring in beer mainly originate from malt (70–80%) and hops [11]. The way raw materials are prepared (refinement of malt), as well as conditions during the processes of mashing and boiling with hops, significantly affect the total polyphenol contents and the degree of the isomerisation of polyphenols in the finished beer product [25]. Polyphenolic compounds have varied chemical structures, which is associated with their diverse capacity for biological activity, including antioxidant activity [33]. The polyphenols contained in beer significantly affect the sensory perceptions of consumers, such as a sense of thickness, a bitter or sour taste, as well as a fullness of flavour. By adding wheat malt (as a part of the input material) it is possible to increase the total polyphenol content in the finished beer product [34]. The total polyphenol contents in wheat beer samples enhanced with sea-buckthorn juice were on average 14.78% (5% *v*/*v* addition) and 6.45% (10% *v*/*v* addition) higher compared to control samples E0 and L0 (Table 3). Sea-buckthorn berries are found with total polyphenol contents in the range of 128–490 mg ·100 g−1 depending on the variety and timing of the harvest [13,18]. The content of polyphenolic compounds is significantly affected by the methods applied during juice production, or more specifically by the process of fruit crushing (breaking cell walls), as well as the thermal processes used to preserve the finished product [23]. Nardini and Garaguso [10] reported total polyphenol contents of 639 mg GAE/L and 399 mg GAE/L, respectively, in beer produced with the addition of orange peel and in apple beer (GAE equivalent of gallic acid). Gasi ´nski et al. [23] investigated beer with the addition of mangoes and reported slightly lower polyphenol contents in the range of 218.6–267.6 mg GAE/L. The addition of persimmon juice led to a decrease in the total polyphenol contents in the beer samples from 433.32 mg GAE/L (25% juice addition) to 290.34 mg GAE/L (75% juice addition [11]). Portuguese commercial fruit beers flavoured with lemon were found with total polyphenol contents in the range of 240–304 mg GAE/L [32].

*Molecules* **2022**, *27*, 3916

**Table 3.** Contents of polyphenols and polyphenolic profiles identified by UPLC-PDA-TQD-MS in wheat beer.

juice. Q-quercetin; K—kaempferol; glc—glucoside; rut—rutinoside; gluc—glucuronide t.c.—trace content below LOQ; GAE—equivalent of gallic acid (mg GAE/L).

Polyphenolic compounds in samples of wheat beer enhanced with sea-buckthorn juice were identified based on an analysis of characteristic spectral data: the mass-tocharge ratio m/z and the maximum absorption of radiation. The characteristics of six polyphenolic compounds that were identified are shown in Table 3. All the identified compounds were flavonols, represented by derivatives of kaempferol and quercetin (in glycoside form). Flavone glycosides are known to have strong antineoplastic and antioxidant properties; they are beneficial for patients with cardiovascular disease and transplants [18]. Kaempferol and quercetin glycosides produce a pungent taste in the mouth, and—to a lesser extent—contribute to a bitter taste, which affects the sensory properties of the finished beer product [18]. The flavonol contents in the control wheat beer samples (E0 and L0) were in the range of 2.18–3.15 mg/L, whereas the addition of defatted sea-buckthorn juice led to an increase in the polyphenol concentrations in the finished beer product by an average of 24.18% and by 48.85% in samples with sea-buckthorn juice added at a rate of 5% *v*/*v* and 10% *v*/*v*, respectively (Table 3). The control wheat beer samples (E0 and L0) were found to contain three compounds, i.e., K-3-*O*-sophoroside, K-3-*O*-rutinoside-7-*O*-glucoside, and K-3-*O*-glucoside-7-*O*-glucoside, which possibly originated from the hops added to the wort during the boiling process; their mean contents were 0.79 mg/L, 0.83 mg/L, and 1.06 mg/L, respectively (Table 3; Figures 3–5). The kaempferol-*O*-glucoside contained in hops is extracted even after 30 min of wort boiling (depending on the dose of the wort [35]). The contents of kaempferol in barley beers were on average in the range of 0.10–1.64 mg/L [36,37]. The wheat beer samples enhanced with defatted sea-buckthorn juice were found to contain the compounds Q-3-*O*-rutinoside-7-*O*-glucoside, K-3-*O*-glucoside, and K-3-*O*-glucuronide-7-*O*-glucoside (Figures 3–5), which were extracted from sea-buckthorn juice during fermentation and may have been rearranged into more complex glycoside derivatives; notably, the concentration of the latter compound was on average two times higher in the wheat beer samples enhanced with sea-buckthorn juice at a rate of 10% *v*/*v* compared to the samples with a 5% *v*/*v* addition of sea-buckthorn juice (Table 3). A study by Guo et al. [38], investigating the fruit of four sea-buckthorn subvarieties, showed the mean contents of Q-3-*O*-rutinoside and Q-3-*O*-glucoside of 32.9 mg·100 g−1 d.w. and 39.7 mg·100 g−1 d.w., respectively. On the other hand, Chen et al. [39] reported the following mean contents of these polyphenols: Q-3-*O*-rutinoside–52.0 mg·100 g−1 d.w. and Q-3-*O*-glucoside–53.3 mg·100 g−1 d.w. (d.w.–dry weight).

**Figure 3.** The chemical formula of identified polyphenols—from left—Q‐3‐*O*‐rut‐7‐*O*‐glc and K‐3‐ *O*‐glc (source: lgcstandards.com; accessed on 14 June 2022). **Figure 3.** The chemical formula of identified polyphenols—from left—Q-3-*O*-rut-7-*O*-glc and K-3-*O*glc (source: lgcstandards.com; accessed on 14 June 2022).

*Molecules* **2022**, *27*, x. https://doi.org/10.3390/xxxxx www.mdpi.com/journal/molecules

**Figure 4.** The chemical formula of identified polyphenols—from left—K‐3‐*O*‐sophoroside and K‐3‐ **Figure 4.** The chemical formula of identified polyphenols—from left—K-3-*O*-sophoroside and K-3-*O*rut-7-*O*-glc (source: lgcstandards.com; accessed on 14 June 2022).

*O*‐rut‐7‐*O*‐glc (source: lgcstandards.com; accessed on 14 June 2022).

**Figure 5.** The chemical formula of identified polyphenols—from left—K‐3‐O‐glc‐7‐O‐glc and K‐3‐ O‐gluc‐7‐O‐glc (source: lgcstandards.com; accessed on 14 June 2022). **Figure 5.** The chemical formula of identified polyphenols—from left—K-3-O-glc-7-O-glc and K-3-Ogluc-7-O-glc (source: lgcstandards.com; accessed on 14 June 2022).

#### *2.3. Sensory Analysis of Wheat Beers*

**Table 4.** Sensory analysis of wheat beer.

Aroma 4.23 ab ±

Taste 3.73 a ±

Foam stability 3.55 a ±

Bitterness 4.00 a ±

Saturation 3.73 a ±

3.86 ab ±

thorn juice; 10—wheat beer with 10% *v*/*v* defatted sea buckthorn juice.

Overall impres‐ sion

formed by a panel of 11 experts are shown in Table 4 and Figures 6 and 7.

*2.3. Sensory Analysis of Wheat Beers* The sensory qualities of the wheat beer samples enhanced with defatted sea‐buck‐ thorn juice determine the specific beer style and contribute to the attractiveness of the beverage for consumers. The results of the sensory assessment of the wheat beers per‐ The sensory qualities of the wheat beer samples enhanced with defatted sea-buckthorn juice determine the specific beer style and contribute to the attractiveness of the beverage for consumers. The results of the sensory assessment of the wheat beers performed by a panel of 11 experts are shown in Table 4 and Figures 6 and 7.

 Mean values within a row with different letters are significantly different (*p* < 0.05). E—'Elixer' variety; L—'Lawina' variety; 0—wheat beer without defatted sea buckthorn juice; 5—wheat beer with 5% *v*/*v* defatted sea buck‐

The wheat beer samples enhanced with defatted sea‐buckthorn juice at a rate of 5% *v*/*v* were found to have the highest sense of flavour (combination of taste and aroma), bitter taste, and saturation, compared to the other beer samples evaluated. With regard to the control beer samples (E0 and L0), the overall impression evaluated by the expert panel was reflected by a score in the range of 3.86–3.91 on a 5‐point scale. The lowest rating was identified in the case of beer samples with a 10% *v*/*v* addition of defatted sea‐buckthorn juice (Table 4). Out of all the quality properties assessed, the stability of the beer head in the wheat beer samples received the lowest rating, whether or not sea‐buckthorn juice was added in the production process. The taste and aroma of the beer are not only affected

**Table 4.** Sensory analysis of wheat beer.

Data are expressed as mean value (*n* = 11) ± SD; SD—standard deviation. Mean values within a row with different letters are significantly different (*p* < 0.05). E—'Elixer' variety; L—'Lawina' variety; 0—wheat beer without defatted sea buckthorn juice; 5—wheat beer with 5% *v*/*v* defatted sea buckthorn juice; 10—wheat beer with 10% *v*/*v* defatted sea buckthorn juice. by the raw materials used but also by the products of the fermentation process (e.g., alde‐ hydes, phenols, and esters), which impact the taste profile of the beer.

*Molecules* **2022**, *27*, x FOR PEER REVIEW 5 of 20

**Figure 6.** Sensory profile of wheat beer—control sample (E0) and sample with defatted sea‐buck‐ thorn juice added at a rate of 5% *v*/*v* (E5) as well as sample with defatted sea‐buckthorn juice added at a rate of 10% *v*/*v* (E10). (\* marks the attributes that were statistically different at *p* ≤ 0.05). **Figure 6.** Sensory profile of wheat beer—control sample (E0) and sample with defatted sea-buckthorn juice added at a rate of 5% *v*/*v* (E5) as well as sample with defatted sea-buckthorn juice added at a rate of 10% *v*/*v* (E10). (\* marks the attributes that were statistically different at *p* ≤ 0.05).

**Figure 7.** Sensory profile of wheat beer—control sample (L0) and sample with defatted sea‐buck‐ thorn juice added at a rate of 5% *v*/*v* (L5) as well as sample with defatted sea‐buckthorn juice added at a rate of 10% *v*/*v* (L10). (\* marks the attributes that were statistically different at *p* ≤ 0.05). **Figure 7.** Sensory profile of wheat beer—control sample (L0) and sample with defatted sea-buckthorn juice added at a rate of 5% *v*/*v* (L5) as well as sample with defatted sea-buckthorn juice added at a rate of 10% *v*/*v* (L10). (\* marks the attributes that were statistically different at *p* ≤ 0.05).

The sensory profile of the investigated wheat beer varied; the control samples (with no addition of sea‐buckthorn juice) had a grainy and malty flavour produced by such

Grains of two varieties of winter wheat, i.e., 'Lawina' and 'Elixer', that were used in the production of wheat beer were obtained from a field experiment conducted in 2021 in the village of Kosina (50°04′17″ N 22°19′46″ E), Podkarpackie Region, Poland. Grain of the winter wheat varieties was harvested after achieving full maturity, and following a resting period, it was used for preparing five‐day wheat malts (the methodology of the malting process was described in Belcar et al. [42]). The wheat malt of the 'Elixer' variety had the following characteristics: extract potential—86.0% d.m., total protein content—11.6% d.m., content of soluble protein—4.71% d.m., diastatic power—331 WK, and degree of final attenuation—82.4%, whereas the wheat malt of the 'Lawina' variety had the follow‐ ing characteristics: extract potential—85.1% d.m., total protein content—11.0% d.m., con‐ tent of soluble protein—4.42% d.m. (d.m.—dry matter), diastatic power—336 WK, and

samples enhanced with sea‐buckthorn juice had a stronger flavour and more refreshing quality as well as an acidic taste, which was more pronounced in the wheat beer samples with a higher proportional share of the juice added. According to the assessing experts, the control samples E10 and L10 would not be accepted by consumers because of their highly acidic taste. The assessing panel expressed an opinion that, irrespective of the wheat variety used to produce the malt, the beer samples had a balanced taste and aroma profile, whether or not sea‐buckthorn juice was added during the production process. The interactions taking place during the fermentation and maturation of beer between esters, sulphur compounds, carbonyls, phenolic compounds, alcohols, and organic acids signifi‐ cantly affect the taste of the produced beer [40]. Beer with a distinct fruity flavour, sweet aftertaste, and pleasant aroma is more favoured and desirable for consumers compared

to traditional types of beer [24,41].

degree of final attenuation—81.7%.

**3. Materials and Methods**

*3.1. Material*

The wheat beer samples enhanced with defatted sea-buckthorn juice at a rate of 5% *v*/*v* were found to have the highest sense of flavour (combination of taste and aroma), bitter taste, and saturation, compared to the other beer samples evaluated. With regard to the control beer samples (E0 and L0), the overall impression evaluated by the expert panel was reflected by a score in the range of 3.86–3.91 on a 5-point scale. The lowest rating was identified in the case of beer samples with a 10% *v*/*v* addition of defatted sea-buckthorn juice (Table 4). Out of all the quality properties assessed, the stability of the beer head in the wheat beer samples received the lowest rating, whether or not sea-buckthorn juice was added in the production process. The taste and aroma of the beer are not only affected by the raw materials used but also by the products of the fermentation process (e.g., aldehydes, phenols, and esters), which impact the taste profile of the beer.

The sensory profile of the investigated wheat beer varied; the control samples (with no addition of sea-buckthorn juice) had a grainy and malty flavour produced by such compounds as maltol and furaneol [40]; they also had a refreshing and sweet taste, characteristic for wheat beers (Figures 6 and 7). The sensory assessment showed that the beer samples enhanced with sea-buckthorn juice had a stronger flavour and more refreshing quality as well as an acidic taste, which was more pronounced in the wheat beer samples with a higher proportional share of the juice added. According to the assessing experts, the control samples E10 and L10 would not be accepted by consumers because of their highly acidic taste. The assessing panel expressed an opinion that, irrespective of the wheat variety used to produce the malt, the beer samples had a balanced taste and aroma profile, whether or not sea-buckthorn juice was added during the production process. The interactions taking place during the fermentation and maturation of beer between esters, sulphur compounds, carbonyls, phenolic compounds, alcohols, and organic acids significantly affect the taste of the produced beer [40]. Beer with a distinct fruity flavour, sweet aftertaste, and pleasant aroma is more favoured and desirable for consumers compared to traditional types of beer [24,41].

#### **3. Materials and Methods**

#### *3.1. Material*

Grains of two varieties of winter wheat, i.e., 'Lawina' and 'Elixer', that were used in the production of wheat beer were obtained from a field experiment conducted in 2021 in the village of Kosina (50◦04017" N 22◦19046" E), Podkarpackie Region, Poland. Grain of the winter wheat varieties was harvested after achieving full maturity, and following a resting period, it was used for preparing five-day wheat malts (the methodology of the malting process was described in Belcar et al. [42]). The wheat malt of the 'Elixer' variety had the following characteristics: extract potential—86.0% d.m., total protein content—11.6% d.m., content of soluble protein—4.71% d.m., diastatic power—331 WK, and degree of final attenuation—82.4%, whereas the wheat malt of the 'Lawina' variety had the following characteristics: extract potential—85.1% d.m., total protein content—11.0% d.m., content of soluble protein—4.42% d.m. (d.m.—dry matter), diastatic power—336 WK, and degree of final attenuation—81.7%.

Materials used in the production of beer samples included commercially available barley malt acquired from Viking Malt company (Strzegom, Poland). The barley malt had the following characteristics: extract potential—80.0% d.m., total protein content—11.4% d.m., content of soluble protein—3.75% d.m. (d.m.—dry matter), diastatic power—324 WK, and degree of final attenuation—82.1%. The wheat and barley malts were refined to the required particle size using a Cemotec disc mill manufactured by FOSS. The input material used in the brewing process comprised commercial barley malt at a rate of 60% and wheat malt at a rate of 40%.

Wheat beer samples were enhanced using defatted juice made from sea-buckthorn berries (after sedimentation) that was produced in 2021 by the Szarłat Company (Cibory Gałeckie, Podlaskie Region, Poland).  Production of Beer*

The production process, based on the infusion method, was carried out in the laboratory of the Department of Agricultural and Food Engineering at the University of Rzeszów. Barley malt with a weight of 3.0 kg and wheat malt with a weight of 2.0 kg were refined and placed in a brew kettle ROYAL RCBM-40N (Expondo; Poland; applied at 80% process efficiency) with 15.0 L of water (3 L of water per each kg of malt). The processes of mashing, boiling with hops, and chilling of beer wort were conducted in line with the methodology described by Gorzelany et al. [8].

All six beer wort samples were found with an extract content of 12.0 ◦P. The chilled wort samples were poured into 30 L fermentation vessels along with the yeast *Saccharomyces cerevisae* Fermentis Safale US-05 (6 × 109/g), earlier subjected to a dehydration process in line with the manufacturer's instructions (0.58 g d.m./L of wort). The fermentation process was carried out at 21 ◦C. After the fermentation process had continued for 7 days, defatted sea-buckthorn juice was added to the beer in specified quantities (0, 5, or 10% relative to wort volume) and then the fermentation process continued for the next 14 days. After 21 days, an aqueous solution of sucrose (0.3%) was added and the beer was poured into bottles for refermentation to achieve an adequate level of carbonation. The beer was then kept at 20 ◦C. Sensory assessment and physicochemical tests were performed one month after the bottling.

Wheat beer produced using malt obtained from the winter wheat variety 'Elixer' and with no addition of defatted sea-buckthorn juice is marked E0, whereas the sample with the 5% *v*/*v* addition of defatted sea-buckthorn juice is marked E5 and the sample with the 10% *v*/*v* addition of sea-buckthorn juice is marked E10. Wheat beer produced using malt obtained from the winter wheat variety 'Lawina' and with no addition of defatted sea-buckthorn juice is marked L0, whereas the sample with the 5% *v*/*v* addition of defatted sea-buckthorn juice is marked L5 and the sample with the 10% *v*/*v* addition of sea-buckthorn juice is marked L10. A total of six variants of wheat beer were produced.

#### *3.3. Analysis of Beer Quality Indicators*

Alcohol contents [% m/m and % *v*/*v*], apparent extract [% m/m], real extract [% m/m], original extract in beer [% m/m], degree of final apparent and real fermentation [%], total acidity [0.1 M NaOH/100 mL], pH, colour [EBC units], carbon dioxide contents [%], contents of bitter substances [IBU units], as well as energy value of beer [kcal/100 mL] were determined following the methodology described by Belcar et al. [34]. The analyses were performed in three replications.

#### *3.4. Contents of Bioactive Compounds in Wheat Beers*

The total contents of polyphenols [mg GAE/L] determined using the Folin–Ciocalteu method, as well as the polyphenol profile of the beer samples, were measured in compliance with the methodology described by Gorzelany et al. [8].

Determination of polyphenolic compounds [mg/L] was carried out using the UPLC equipped with a binary pump, column and sample manager, photodiode array detector (PDA), tandem quadrupole mass spectrometer (TQD) with an electrospray ionization (ESI) source working in negative mode (Waters, Milford, MA, USA) according to the method of Zurek et al. [ ˙ 43]. Separation was performed using the UPLC BEH C18 column (1.7 µm, 100 mm × 2.1 mm, Waters) at 50 ◦C at a flow rate of 0.35 mL/min. The injection volume of the samples was 5 µL. The mobile phase consisted of water (solvent A) and 40% acetonitrile in water *v*/*v* (solvent B). The following TQD parameters were used: capillary voltage of 3500 V; con voltage of 30 V; con gas flow 100 L/h; source temperature 120 ◦C; desolvation temperature 350 ◦C; and desolvation gas flow rate of 800 L/h. Polyphenolic identification and quantitative analyses were performed based on the mass-to-charge ratio, retention time, specific PDA spectra, fragment ions, and comparison of data obtained with commercial standards and literature findings. The analyses were performed in three replications.

Contents of ascorbic acid [mg/100 mL] in sea-buckthorn berries were assessed in conformity with PN-A-04019:1998 [44]. The analyses were performed in three replications.

#### *3.5. Antioxidant Activity in Wheat Beers*

Antioxidant capacity of fruit beers (assessed using DPPH [mM TE/L], FRAP [mM Fe2+/L, and ABTS [mM TE/L] assays) was measured following the methodology described by Gorzelany et al. [8]. The analyses were performed in three replications.

#### *3.6. Sensory Assessment in Beers*

Sensory assessment was performed by a panel of 11 experts (4 women and 7 men, aged 30–40 years), in a sensory analysis laboratory in line with the EBC 13.13 method [45]. Beer samples, chilled to a temperature of 10 ◦C and coded, were served in a random order in transparent plastic cups with a capacity of 250 mL. After each test, the experts were given water to rinse their mouths. Sensory analysis of the beer samples was performed using a 5-point scale, assessing the specific quality characteristics, i.e., aroma (5—very strong, distinctive, and pleasant; 1—imperceptible aroma/ unpleasant smell), taste (5—very good; 1—bad); beer head stability (5—highly stable; 1—unstable), bitter taste (5—weak; 1—very strong), and carbonation (5—high; 1—poor or none). The average score described the general impression (5—excellent; 1—poor) related to the investigated wheat beers. Additionally, evaluation of the beer samples in terms of their taste and aroma applied the sensory profile describing the quality characteristics (malty, fruity, sweet, grainy, strong, full, fresh, phenolic, bitter, and sour) in line with EBC 13.12 [46]. The sensory profile of the fruit beer produced with the addition of defatted sea-buckthorn juice was compared to the control beer (no addition of sea-buckthorn juice).

#### *3.7. Statistical Analysis*

The results of the fruit beer evaluation are shown as mean values and standard deviations. The statistical analyses of the results were computed using Statistica 13.3 (TIBCO Software Inc., Tulsa, OK, USA). The results related to physicochemical characteristics, polyphenol contents, and antioxidant activity of fruit beer samples were examined using the two-factor completely randomized ANOVA with a significance level of α = 0.05. The mean values were compared using the Tukey HSD test.

#### **4. Conclusions**

The study, designed to assess the feasibility of defatted sea-buckthorn juice as an enhancer to be used in the production of fruit wheat beers, showed that the most balanced sensory profile (intensity, perceived bitter flavour, as well as fruity taste and aroma) is found in beer samples enhanced with juice at a rate of 5% *v*/*v*. Additionally, these beer samples were shown to have better colour, as well as higher polyphenol contents and antioxidant capacity. Although the addition of defatted sea-buckthorn juice at a rate of 10% *v*/*v* positively affected the health-promoting properties of wheat beer (with a mean content of ascorbic acid of 4.5 mg/100 mL), the sensory properties of this type of beer were not acceptable for the assessing panel, mainly due to its highly acidic taste. The enhancement of wheat beer with defatted sea-buckthorn juice at a rate of 5% *v*/*v* could effectively be applied to expand the assortment of fruit wheat beers on offer.

**Author Contributions:** Conceptualization, J.B. and J.G.; methodology, J.B.; validation, J.B. and J.G.; formal analysis, J.B.; data curation, J.B.; writing—original draft preparation, J.B.; writing—review and editing, J.B. and J.G.; visualization, J.B.; supervision, J.G.; project administration, J.B.; funding acquisition, J.B. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

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

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** The authors wish to express their gratitude to the M. and W. Lemkiewicz 'Szarłat' company from Cibory Gałeckie (Podlaskie Region, Poland) for the material provided for the present study (defatted sea buckthorn juice).

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

**Sample Availability:** Samples of the compounds used in the research are available from the authors.

#### **References**

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**7. Application of PRP in the treatment of chronic wounds**

The process of healing of skin wounds is very intense and includes a number of phenomena, such as hemostasis, inflammation, formation of granulation tissue, epithelialization, neovas‐ cularization, collagen synthesis, and wound contraction. Several studies demonstrate that the growth factors are depleted in chronic wounds. Platelets have a tendency to aggregate when activated, having a leading role in skin healing. Activated platelets release growth factors, adhesion molecules, and lipids through which migration, proliferation, and the function of keratinocytes, fibroblasts, and endothelial cells are regulated [47].

ished of platelets. The macrophages clean the area through phagocytosis and secrete factors responsible for the formation of granulation tissue from the fibroblasts. During this phase, angiogenesis also begins under the influence of growth factors and thrombin. With the recovery of vascular endothelial cells new vessels also develop. Endothelial cells are activated by thrombin, while a process of interaction is also ensured, which restricts the intensity of the formation of new vessels [40]. In this phase mesenchymal stem cells also emerge and their differentiation is performed in specific tissues such as bone, cartilage, or vascular tissues being

At the time of the remodeling phase, contraction of the collagen and convergence of the edges of the wound is observed. Cell density and vascularization is reduced, excess matrix is removed, and the collagen fibrils are aligned along the stress lines, which increases the strength of the newly-formed tissue [10]. Accumulated granulation tissue remodels or slowly trans‐

A number of growth factors are located in platelet alpha-granules. In order to be released into the injured tissue monocytes, neutrophils, fibroblasts, mesenchymal stem cells and osteoblasts need the chemotactic effect of an available growth factor. Platelet-derived growth factor (PDGF) produces such effect. This growth factor influences mitogenesis of fibroblasts and smooth muscle cells. PDGF improves the formation of fibrous tissue, assists in the three phases of the healing cascade, and also significantly affects angiogenesis and re-epithelialization. Another important growth factor is transforming growth factor beta (TGF-b). It impacts the connection between fibronectins. It influences cell migration, proliferation and replication, and

Chronic wounds' and endochondral ossification's healing processes are highly influenced by vascular endothelial growth factor (VEGF). It is active in angiogenesis, too [13,42]. The next factor taking part in the recovery of chronic wounds is epidermal growth factor (EGF). It also influences the mitogenesis of endothelial cells, fibroblasts, and keratinocytes [42]. Hepatocite growth factor (HGF) is located in various tissues (several types of epithelium, liver, lung, kidney and tumor). It affects tissue regeneration and possesses morphogenic, mitogenic,

The process of healing of skin wounds is very intense and includes a number of phenomena, such as hemostasis, inflammation, formation of granulation tissue, epithelialization, neovas‐ cularization, collagen synthesis, and wound contraction. Several studies demonstrate that the growth factors are depleted in chronic wounds. Platelets have a tendency to aggregate when activated, having a leading role in skin healing. Activated platelets release growth factors,

determined by chemical signals [36,38,39].

is present during inflammation processes [41].

antiapoptotic, and neurotrophic qualities [38,43-46].

**7. Application of PRP in the treatment of chronic wounds**

**6. Platelet growth factors**

178 Immunopathology and Immunomodulation

forms into a specific tissue such as skin or bone [36,38,39].

Skin wounds are defined as the absence of tissue, where epidermis and dermis are affected, while sometimes can reach the adipose tissue and muscle fascia. In this process, no natural regeneration is observed and tissue lesion is transformed into fibrotic scarring [48,49]. Skin wounds have a frequency of 0.78% and huge funts are spent for their treatment. Annually, 2% of the budget is used for health in the European Union (EU), £ 40 million in the UK, \$-1.3 billion are used for the treatment of decubitus in the US [48].

According to Zhu et al., chronic cutaneous wounds are quite frequent in developing countries. They are difficult to treat due to insufficient amount of growth factors. Sometimes wound infections are observed. Conventional treatment with various known techniques and methods is not enough due to the insufficiency of growth factors. Dressing and surgical debridement is used for these patients, but these strategies do not lead to significant outcomes [50].

For the development of skin wounds facilitate various etiological factors such as: chronic venous disease, peripheral arterial disease, neuropathy, arterial hypertension, physical injury, hematologic disorders, skin infection, inflammatory diseases, neoplasms, iatrogenic changes, and those associated with nutrition [48,49,51].

PRP gel has been used for wound healing stimulation on molecular and cellular levels for the last two decades [48,52]. Martinez-Zapata et al. showed tissue regeneration in maxillofacial surgery, chronic ulcers, and surgical wounds in their systematic review [53]. Weglein et al. assessed studies dealing with diabetic wound healing and another study reviewed the healing of chronic wounds [24]. Carter et al. reviewed wound healing when PRP gel is applied and wound healing of control groups (conventional wound care) and reported on a number of indicators [48,52].

With these studies, it was observed that PRPs have effectively stimulated healing in wounds with delayed healing. In a study, Martinez-Zapata et al. reported that the percentage of full healing of wounds treated with PRP has increased compared to that of control wounds. Carter et al. performed a meta-analysis of articles for chronic wounds and support the idea that the use of platelet rich plasma favors full healing compared to control groups [9,19,43]. In a study by Villela et al., they reached similar conclusions, with one of their conclusions reaching so far as to conclude that PRP is the method of choice for the treatment of wounds [48,21].

Difficult to heal wounds are considered those that do not heal after the fourth week of their appearance after being treated with standard methods for the particular pathology. Chronic is a wound that is not healing for a period of three months. The evaluation of each wound is very important. For the purpose, a number of point scales suggested by Cancela et al. based on clinical and anatomical criteria for the size of the wound are used, while a patient variable is also taken into account. The points are randomly defined using clinical experience in wound healing. Common parameters are assessed such as the presence or absence of erythema and edema surrounding the wound, purulent discharge, presence of fibrin, swelling in pressure, swelling with ocher dermatitis, and granulation [48].

Anatomical features are noted and graded such as open bone or tendon, location of the wound, and quality of the pulse of artery dorsalis pedis and artery tibialis posterior (when related to wound location) [48]. Wounds are measured to determine their overall surface, depth, and distribution of undermining of the wound edges. The duration of the wound is determined by the history [48].

There are many studies relating to the pathophysiology of wounds. Skin wound healing is an intensive process and in it are observed a number of events such as hemostasis, inflammation, formation of granulation tissue, formation of new vessels, collagen synthesis, epithelialization, and contraction of the wound. The process of reduction of growth factors in chronic wounds is demonstrated in numerous reports as a result of their reduced production and release, sequestration, or degradation. These mechanisms can be combined [48,54].

Platelet aggregation has a clearly expressed effect in the process of skin wound healing. In this process, growth factors are released and, adhesion of molecules and lipids is observed, while they are responsible for the regulation of cellular migration, proliferation and function of keratinocytes, fibroblasts, and endothelial cells [11,55]. Platelets secrete several antimicrobial peptides when activated by thrombin. PRP has an important antimicrobial and immunoregu‐ latory activity obtained from leukocytes [56,57]. A review of studies using different products rich in platelets demonstrates significant improvement in the treatment of chronic wounds.

Anitua et al. presented an open and randomized study to evaluate the effect of PRP in chronic ulcers of 14 patients. They reported good healing response in 80% of the cases after 8 weeks of treatment, compared with 20% in the control group. Leukocytes were not detected in the analyzed products. Their explanation for the good results lies in the high concentrations of growth factors [48,54].

Crovetti et al. monitored the evolution of chronic skin wounds in 24 patients treated with autologous or homologous gel (depending on the case) and observed a complete healing in 9 of them after an average of 10 applications, with a reduction of pain in all cases [48,49].

Marté-Mestre et al. reported the recovery of vascular chronic ulcers with the use of PRP in 12 of 14 patients for an average treatment period of 2.93 months (average of 0.5 - 7 months) [48,58].

Dellinger and Britton applied autologous platelet gel and reported positive results, with no complications and acceleration of the healing process (5 - 8 weeks) regardless of Pan size. A reduction of the risk of amputation and improvement of quality of life was observed [22,48]. PRP has the advantage of having no adverse effects [59].

Margolis et al. found in a retrospective group study with neuropathic wounds in diabetic feet greater efficiency in the use of PRP against the conventional therapies, with a more obvious effect in severe wounds [48,60].

PRP may be used for treatment of various chronic cutaneous wounds, especially when standard conventional therapy is not good enough and surgical treatment is not possible. It reduces the duration, cost of treatment and the hospital stay. These procedures may be performed in outpatient clinics. There is reduction of wound pain after starting the treatment, reduced risk of blood-borne disease transmission, wound healing is restored, and local immunity is activated [61,62].

#### **8. PRP in osteoarthritis**

Anatomical features are noted and graded such as open bone or tendon, location of the wound, and quality of the pulse of artery dorsalis pedis and artery tibialis posterior (when related to wound location) [48]. Wounds are measured to determine their overall surface, depth, and distribution of undermining of the wound edges. The duration of the wound is determined

There are many studies relating to the pathophysiology of wounds. Skin wound healing is an intensive process and in it are observed a number of events such as hemostasis, inflammation, formation of granulation tissue, formation of new vessels, collagen synthesis, epithelialization, and contraction of the wound. The process of reduction of growth factors in chronic wounds is demonstrated in numerous reports as a result of their reduced production and release,

Platelet aggregation has a clearly expressed effect in the process of skin wound healing. In this process, growth factors are released and, adhesion of molecules and lipids is observed, while they are responsible for the regulation of cellular migration, proliferation and function of keratinocytes, fibroblasts, and endothelial cells [11,55]. Platelets secrete several antimicrobial peptides when activated by thrombin. PRP has an important antimicrobial and immunoregu‐ latory activity obtained from leukocytes [56,57]. A review of studies using different products rich in platelets demonstrates significant improvement in the treatment of chronic wounds.

Anitua et al. presented an open and randomized study to evaluate the effect of PRP in chronic ulcers of 14 patients. They reported good healing response in 80% of the cases after 8 weeks of treatment, compared with 20% in the control group. Leukocytes were not detected in the analyzed products. Their explanation for the good results lies in the high concentrations of

Crovetti et al. monitored the evolution of chronic skin wounds in 24 patients treated with autologous or homologous gel (depending on the case) and observed a complete healing in 9 of them after an average of 10 applications, with a reduction of pain in all cases [48,49].

Marté-Mestre et al. reported the recovery of vascular chronic ulcers with the use of PRP in 12 of 14 patients for an average treatment period of 2.93 months (average of 0.5 - 7 months) [48,58].

Dellinger and Britton applied autologous platelet gel and reported positive results, with no complications and acceleration of the healing process (5 - 8 weeks) regardless of Pan size. A reduction of the risk of amputation and improvement of quality of life was observed [22,48].

Margolis et al. found in a retrospective group study with neuropathic wounds in diabetic feet greater efficiency in the use of PRP against the conventional therapies, with a more obvious

PRP may be used for treatment of various chronic cutaneous wounds, especially when standard conventional therapy is not good enough and surgical treatment is not possible. It reduces the duration, cost of treatment and the hospital stay. These procedures may be performed in outpatient clinics. There is reduction of wound pain after starting the treatment,

PRP has the advantage of having no adverse effects [59].

sequestration, or degradation. These mechanisms can be combined [48,54].

by the history [48].

180 Immunopathology and Immunomodulation

growth factors [48,54].

effect in severe wounds [48,60].

Cartilage defects and the most common joint disease, osteoarthritis(OA), are characterized by degeneration of the articular cartilage that ultimately leads to joint destruction [50]. Several treatments for OA exist, including exercise, weight control, bracing, nonsteroidal antiinflammatories, cortisone shots, and viscosupplementation. The biochemical environment of the disease process should be influenced in order to achieve better control of the symptoms. There is an imbalance between various cytokines resulting in the increased quantity of proteolytic enzymes leading to cartilage destruction [25,63 - 65]. The repair of damaged articular cartilage and bone defects represents a great challenge for the orthopedist because of the regenerative limitations of cartilage and the difficulty in obtaining an adequate bone substitute. Damages to the cartilage havess been treated by microfracture, debridement, and grafting procedures [42]. The results obtained by these techniques are so far unsatisfactory, and in most cases, these techniques result in deposition of fibrous connective tissue with low mechanical strength at the defect location [56,66]. Broad application of PRP is typical for oral, maxillofacial, and plastic surgery, while in the last few years there have been papers showing administrations in orthopedics and traumatology. When obtaining PRP, high concentration of growth factors in a volume unit is observed and acceleration of the regeneration process in the arthritic cartilage. Consecutively, PRP stimulates local cell activity. This technique is lowpriced and has no side-effects or dangers for the patient [33,67].

The presence of concentrated growth factors modulates the phenotypic expression of chon‐ drocytes. This improves their recovery. TGF-b plays a major role in the differentiation of stem cells into chondrocytes. Synthesis of cartilaginous matrix is improved, as well. PDGF and IGF are both key parts of the recovery process, as the first one stimulates proliferation of chondro‐ cytes while the second one increases the synthesis of proteoglycan - macromolecules. Thus, the strength of cartilage is improved [30,56]. Not only does PRP stimulate cellular activity and the process of regeneration and repair, but it also improves bone and cartilage recovery when biomaterials are applied [68,69]. In order to recover cartilage defects, not only PRP is needed but also an adequate biomaterial that has similar characteristics as the natural tissue and promotes cellular adhesion, proliferation, and differentiation [56].

According to Hunziker et al., TGF-b increases chondrogenesis [24,70], while Kon et al. documented PRP amplification of chondrocyte proliferation with convincing clinical effects on degenerative knee OA [24,25,70,71]. Results of the Kon et al. publication on 115 arthritic knees treated with PRP showed improvement in the functional status and pain scores "which remained positive at 6 months, with only mild degradation of scores at 1 year". It was also mentioned that lower grades of arthritis and younger patients had better outcomes [25,72].

Qi et al. shared results on the treatment of osteochondral defects in rabbits with PRP and biodegradable collagen scaffolds. PRP was put into collagen scaffolds (0.05 ml PRP/structure) and then implanted in osteochondral defects (4 x 3 mm) produced in the patellar sulcus of rabbit femurs. Evaluation of tissues was completed after 6 and 12 weeks by histological analysis and a mechanical indentation test. The results in both follow-up times were improved and showed that PRP possesses the capacity to stimulate the regeneration of critical defects in cartilage [56,73].

According to Patel et al. in a study with level 1 evidence, PRP has a superior effect compared to placebo in patients with varying degrees of knee OA. They tested 78 patients (156 knees) and observed that PRP alleviated the symptoms of OA when compared with a saline injection used as control. Follow-up period was up to 6 months. The results showed that with the help of PRP, the homeostasis and function of the knee joint can be improved [74,75].

Wei et al. reported the effect of PRP on the regeneration of cartilage in vivo. Auricular cartilage chondrocytes from rabbits were added to PRP (5 x 107 cells/ml PRP), injected subcutaneously in the dorsum of the donor animal, and after 60 days the tissue was evaluated by magnetic resonance, histological, and histochemical analysis. The results exhibited cartilaginous tissue formation in those groups that were injected with chondrocytes and PRP. In the groups in which only PRP was injected, cartilaginous tissue formation was not

observed. The authors inferred that PRP is effective in stimulating regeneration of cartilage when associated with viable cells [56,76].

A level 1 study published in 2012 in the Journal of Arthroscopy by Sanchez et al. used PRP type 4A to observe the results of PRP injection compared with hyaluronic acid. In this study 176 patients, were randomized to receive three weekly applications of PRP (8 ml at weekly intervals) or hyaluronic acid injections. The study was well-conducted, but the results only focused on the short term (until 6 months of follow up). The study was awarded as the best randomized clinical trial (level 1) for 2012, which gives useful fundamental guidelines on how to study the effects of PRP [74,77,78].

In the light of orthopedic and trauma critical literature, there is no standardization of treatment methods, such that most of the works use 3–5 applications of PRP in intervals ranging from 1–3 weeks with no standardization of the amount of volume to be applied and the ideal platelet concentration method [74].

Important factors for results of PRP treatment are the amount of platelets, grade of chondrocyte damage, patient's physical activity, and medical history combined with psychosocial factors. It is good to investigate whether PRP balances anabolism-catabolism, and if there is, what effect of does the PRP have on the synovium. Given the patient's condition, appropriate PRP treatment in OA should use more anti-inflammatory cytokines instead of pro-inflammatory cytokines. The future of OA research aims at isolating cytokines contained in PRP termed as "autologous conditioned serum" to specifically inhibit IL-1 and TNF alpha and down regulate MMP-13 [25,79].

Early OA can be treated by implementing intra-articular PRP. Worse outcome is observed with patients of advanced age and disease process. Khoshbin et al. compared intra-articular administration of PRP to hyaluronic acid and non-setoroid injections and reported that sequential intra-articular PRP injections may have beneficial effects on adult patients with mild to moderate osteoarthritis for a period of 6 months [80].

Zhu et al. reported the fact that PRP contains growth factors, which may have a negative impact on the treatment of a joint with osteoarthritis. That is why those growth factors should be isolated. The following years may be focused on finding suitable and harmless growth factors to improve PRP outcome [50]. Dold et al. paid attention to the fact that there is insufficient data on PRP implementation on post-traumatic osteochondral defects. Further studies with longterm follow-up are necessary in order to illustrate the benefits of isolated PRP application or application in combination with surgical treatment when treating cartilage pathology [81].


2.2.4. Three-Item Cognitive Reflection Test

After having completed the above-mentioned tasks, the subjects proceed to the three-item CRT [5]. The three-item CRT can be found in Appendix B.

The test is used to detect an individual's proclivity for applying two systems of decision-making: System 1 and System 2 processes [19]. System 1 is the intuitive "part" of the brain that relies on heuristics and automaticity. It possesses no computational capacity and is characterized as unconscious. It is fast, automatic and requires no effort. System 2 is the more analytical and rational system. It is deliberate and activated when facing complex calculations, different choices and requires the individual to be focused [20]. The performance on CRT indicates whether an individual is able to overcome the desire to go with the intuitive (incorrect) answer, reflect further upon the question and reach the, when explained to, relatively easy correct answer. For example the first question of the CRT: *A bat and*

1 The experimental design applied actual matching on the subset of subjects who gave DKK 0 in the *dictator game*. Ex ante it could be expected that at least one subject would do so, based on previous *dictator game* experiments (In a meta study [18] found that 36.11% of all participants chose to give nothing).

*a ball cost \$1.10. The bat costs \$1.00 more than the ball. How much does the ball cost? \_\_\_ cents. Intuitive Answer: 10 / Correct Answer: 5.*

Based on the answers to the CRT I divide subjects into three groups using the categorization used by [21]: Subjects who answered correctly two or more items on the CRT are categorized as *reflective*. Those opting for the intuitive, but wrong answer at least in two of the three items are *intuitive*. The subjects who are not categorized as either *reflective* or *intuitive*, form the residual group. For precise details of the categorization, see Appendix C.