Ancient farm,

in the province of Pavia

Effect of winemaking on the composition of red wine. Wine as a source of polyphenols for anti-infective biomaterials

Arianna Di Lorenzo 1, Nora Bloise 2, Silvia Meneghini 1, Antoni Sureda 3, Gian Carlo Tenore 4,

Livia Visai 2,5, Carla Renata Arciola 6,7, * and Maria Daglia 1, *

1. Department of Pharmaceutical Sciences, Pharmaceutical Chemistry and Pharmaceutical Technologies Section,

University of Pavia, V.le Taramelli 12, Pavia 27100, Italy;

2. Department of Molecular Medicine, Health Technologies Center (CHT), UdR INSTM, University of Pavia,

Viale Taramelli 3 / b, Pavia 27100, Italy;

3. Research Group on Community Nutrition and Oxidative Stress and CIBEROBN

(Pathophysiology of Obesity and Nutrition), University of the Balearic Islands, Palma de Mallorca, Balearic Islands

4. Department of Pharmaceutical and Toxicological Chemistry, University of Naples Federico II,

Via D. Montesano 49, Naples 80131, Italy;

5. Department of Occupational Medicine, Toxicology and Environmental Risks, S. Maugeri Foundation, IRCCS,

Via S. Boezio 28, Pavia 27100, Italy

6. Research Unit on Implant Infections, Rizzoli Orthopedic Institute, via di Barbiano 1/10,

Bologna 40136, Italy

7. Department of Experimental, Diagnostic and Specialized Medicine (DIMES), University of Bologna,

via San Giacomo 14, Bologna 40126, Italy

SummaryBiomaterials releasing bactericides have now become medical tools for device-associated infections. The ideal anti-infectious biomaterial is to counteract infection while

safeguard the integrity of eukaryotic cells. Red wine is a widely consumed beverage that has many biological properties attributed to it, including protective effects against oral infections and related bones (osteoarthritis, osteomyelitis, periprosthetic joint infections) and cardiovascular disease. In this study, fifteen samples of red wine derived from native grapes of Oltrepò Pavese (Italy), obtained from the vinification processes of the red wine "Bonarda dell'Oltrepò Pavese", were analyzed alongside three samples obtained by pressing the pomace. Total polyphenol and monomeric anthocyanin content were determined and metabolite profiling was conducted by chromatographic analysis. The antibacterial activity of the wine samples was evaluated against Streptococcus mutans, responsible for dental caries, Streptococcus salivarius and Streptococcus pyogenes, two oral bacterial pathogens. Results highlighted the winemaking phases in which the samples have the highest polyphenol content and the greatest antibacterial activity. Considering the global need for new weapons against bacterial infections and alternatives to conventional antibiotics, as well as the favorable bioactivity of polyphenols, the results point to red wine as a source of antibacterial substances to develop new biomaterials and anti-infective coatings for biomedical devices.

Introduction: Growing evidence suggests that natural antimicrobial substances can be used in a variety of ways such as food products (i.e., food preservatives and active ingredients, smart eco-friendly packaging), cosmetic products (e.g. oral care cosmetics, such as toothpastes and mouthwashes), medical devices and antimicrobial drugs. Regarding the latter issue, infections are the main causes of morbidity and mortality in the world. According to the World Health Organization reports, there were more than 55 million deaths worldwide in 2011 with infection responsible for one third of all deaths. Long-term exposure to antibiotics led to antibiotic resistance of microorganisms; therefore, in recent years, much attention has been paid to the discovery and development of new natural antimicrobial agents that could act against resistant microorganisms.

Considering that biomaterial research is one of the most important and fastest growing fields in modern food science and medicine, natural products, particularly of plant origin, could be new and interesting candidates for biomaterial applications. Several edible antimicrobial films have been produced to minimize the growth of pathogenic microorganisms, including those based on proteins (wheat gluten, casein and whey proteins, polysaccharides (chitosan) and lipids (acylglycerols). In recent years, There has been a growing interest in adding antimicrobial agents to medical devices. The goals of the addition are the reduction or prevention of a device-related infection and the reduction or inhibition of their bacterial colonization. For example, substances antimicrobials (i.e. thyme oil) have been incorporated into chitosan films for potential applications in wound healing.

Plant foods have aroused great interest as a source of antimicrobial substances. Among the secondary metabolites present in plants, polyphenols, terpenoids, alkaloids, lectins, polypeptides and polyacetylenes are known to be antimicrobial agents. Most of these metabolites are safe and show negligible toxicity and side effects. Green tea, cranberry, and cocoa are considered to be the most promising plant sources. Many investigations have shown that grapes and related products (grape juice and wine) led to exert antibacterial activity against a wide range of bacteria due to the low polyphenol effect.

Wine, mainly fermented from Vitis Vinifera L. grapes, is a widely consumed drink due to its pleasant sensory properties. Grapes and their wines are characterized by high levels of polyphenols, a large family of secondary plant metabolites considered responsible for the quality of wine and its positive effects on human health.

The qualitative and quantitative phenolic composition of red wine is influenced by various factors, such as the grape variety, the position of the vineyard, the climatic conditions and sun exposure, the winemaking process and the conservation of the wine.

Red wines, obtained from dark-skinned vines, generally contain up to 3500 mg / L of phenolic substances including non-flavonoids and flavonoids, the latter group which represents an important percentage of phenolic compounds, with quantities between 1000 and 1800 mg / L. The main common representative classes of flavonoids

they are flavonols, dihydroflavonols, anthocyanins, proanthocyanidins and flavan-3-oils. Anthocyanins and proanthocyanidins accumulate mainly in the peel of berries and are responsible for sensory properties (i.e. color, astringency, bitterness and aroma) and for chemical stability against oxidation. Furthermore, tannins are able to form complexes with saliva proteins, giving astringency and structure to the final drink. Non-flavonoid compounds include hydroxycinnamic and hydroxybenzoic acids and phenolic acids which reach concentrations up to 200 mg / L in red wines. Other minor non-flavonoids components are volatile phenols, responsible for unpleasant aromas, and stilbenes, with trans-resveratrol recognized as one of the characteristic components of wine.

Red wine is traditionally obtained from the pressing and destemming of grapes, with the must obtained by remaining in contact with skins and seeds for a longer or shorter period: in this way, the dissolution of the coloring substances of the marc, and possibly of the stalks, is assisted by the presence of alcohol produced during fermentation. During the fermentation process, the wine is moved from one barrel to another (racking), until the end of both alcoholic and malolactic fermentation.

Today traditional winemaking has been improved thanks to technological innovations allowing the production of better quality wines and the automation of processes according to the winemaking method, it is possible to obtain wines with different specific sensorial properties and different quantities of bioactive compounds. The addition of co-pigments before prolonged fermentative maceration, generally hydroxycinnamic acids, hydroxycinnamoyltartaric acids and flavonols, for example, can be used to improve the color stability of the wine and to change its shades from purple to deep red. During maceration, the anthocyanins in wine come into contact with co-pigments, converting from monomers to polymers. Another winemaking procedure used to improve the phenolic content and sensory properties is the addition of condensed tannins derived from grapes. Tannins require an increase in anthocyanin stabilization through the formation of polymer pigments, providing an improvement in the wine's color and antioxidant intensity and radical scavenging properties. Furthermore, a new winemaking technique consists in leaving yeast lees in the wine after alcoholic fermentation. This procedure induces noticeable changes in the wine aroma, because the lees have the ability to adsorb and release some volatile compounds.

Finally, cryomaceration is widely used in the production of white wines and has recently been introduced among the red vinification treatments. This procedure allows a greater extraction of phenolic compounds contained in the skins and of primary aroma. The yield of cold pre-fermentation maceration is affected

from parameters that describe the process itself, such as the working temperature (normally between 5 and 5 C), the use of cryogenic gas (N2 or CO2) instead of the refrigeration units and the duration of the cryomaceration period. Depending on these parameters, cryomacerated red wines usually show a greater aromatic intensity and greater stability in their gustatory properties, a better qualitative profile and a higher content of flavonols (e.g. quercetin), but a lower content of anthocyanins, catechins and resveratrol .

A growing body of evidence suggests that moderate intake of red wine exerts protective effects on human health, strongly related to its polyphenolic composition. Polyphenols are well known antioxidants and free radical scavengers. Furthermore, they can inhibit some transcriptional factors and is able to modulate enzymatic activity and metabolic pathways, thus exerting particular health benefits regarding chronic degenerative diseases such as cardiovascular diseases, cancer and diabetes and its complications (i.e., retinopathy diabetic).

Recent studies show that moderate consumption of red wine is also linked to the protection of oral hygiene wine. In particular, Daglia et al. tested the antibacterial activity of two commercial wines against oral streptococci responsible directly (S. mutans) or indirectly (S. salivarius) for the development of caries and against S. pyogenes responsible for pharyngitis. The proven red and white wines were active against the tested strains. This activity was mainly attributed to the presence of organic acids (i.e. lactic, malic, succinic, tartaric, citric and acetic). Furthermore, the same research group showed the in vitro and ex vivo anticary activity of red wine, focusing on its antibiofilm and antiadhesive properties. Dealcoholised red wine, and in particular its proanthocyanidins, has been shown to strongly interfere with the adhesion of Streptococcus mutans to saliva-coated hydroxyapatite (sHA) spheres, promoting their detachment from sHA and potently inhibiting biofilm formation in vitro. Furthermore, the ability of red wine to ex vivo inhibit S. mutans biofilm formation on the occlusal surface of natural human teeth were also shown. The same results were achieved by more recent research which reported that red wine exerted antimicrobial properties in a supragingival plaque biofilm model, supplemented with five bacterial species (e.g. Actinomyces oris, Fusobacterium nucleatum, Streptococcus oralis, Streptococcus mutans and Veillonella dispar) commonly associated with oral diseases.

In recent decades, numerous investigations have been conducted around the world on diseases of the oral cavity because these diseases (such as dental caries and periodontal diseases) are serious health problems that affect the quality of life and are linked to systemic diseases, such as diseases cardiovascular, rheumatoid arthritis, osteoporosis and osteomyelitis.

Several synthetic compounds are commercially available for oral health and the treatment of dental caries. However, these substances can alter the oral microbiota and are responsible for side effects (e.g. diarrhea and tooth staining). Furthermore, these products induce uncommon infections and antimicrobial resistance, which poses a global threat to global public health due to new resistance mechanisms that continue to emerge, greatly increasing the risk of spreading resistant diseases.

Alternative prevention and treatment options for dental caries should be put in place to further reduce caries around the world, particularly in developing countries, which need new technologies that are safe, effective and cost-effective for disease treatment products. Phytochemicals are a possible source of effective, cheap and safe anticarying agents and appear to be a suitable alternative.

Although there have been changes in the phenolic composition during the previously studied winemaking process, few studies are available to date on monitoring the functional properties of wine during the various stages of winemaking.

Most of the existing studies are related to the evaluation of the antioxidant activity by means of different methods (eg FRAP, DPPH, oxygen radical absorption test (ORAC)). The purpose of this research is to collect combined data on the determination of the chemical composition of wine (metabolic profiling, phenolic and anthocyanic content) and evaluation of its antibacterial properties against oral pathogens during the course of winemaking, starting from the grape pressing until the end of the fermentation. The most promising phase of winemaking, during which the wine has the highest antibacterial activity, will be identified to obtain extracts useful for the preparation of oral health products active against the diseases induced by the tested bacteria.

Wine samples from a 2015 vintage, obtained from Croatina and Barbera V. Vinifera L. grapes grown in the Oltrepò Pavese, were obtained from the Italian Oltrepò winery "I Doria di Montalto" (Montalto Pavese, Pavia, Italy ). The Croatina wine samples were collected during the grape pressing phase (30 September 2015), at each racking into the fermentation tank and at the end of fermentation (30 October 2015).

All wine samples, their date of collection, the winemaking stage and the abbreviation used in this document are listed in table 1.

Croatina wine is obtained through a process conceived and developed by “I Doria di Montalto” winemakers of the winery, consisting of hyper-oxygenation of the must during fermentation and micro-oxygenation during the post-fermentation phases.

Subsequently, 48 h after the start of alcoholic fermentation in vitrified concrete tanks, the temperature is controlled not to exceed 27 C. Then, 7 days after the start of fermentation, the pomace is de-stemmed

from the must and undergoes a softer pressing at 1 atm: the wine obtained, called Croatina Torchiato, was analyzed in three different phases of this vinification process. The Croatina wine is then racked for 5 days, to allow malolactic fermentation due to the Oenococcus Oeni and Leucococcus bacteria.

In conclusion, this work represents the first attempt to investigate changes in the chemical composition of red wine, together with changes in its antibacterial activity, which occur during the winemaking process (including crushing, fermentation and crushing). As far as the chemical composition is concerned, the main variations in the concentration of total and total polyphenols monomeric anthocyanins are positively influenced by alcoholic fermentation and negatively influenced by the addition of SO2. At the end of fermentation the Bonarda red wine reached a TPC and TMA concentration comparable to other red wines in the same vinification phase. Metabolite profiling does not change significantly during the winemaking process and the differences measured can be explained by the various conditions that occur during the process itself. Regarding the antibacterial activity, it is possible to summarize the results described as follows:

(i) all wines harvested during winemaking show an antibacterial effect against the bacterial strains tested;

(ii) the effect can be considered strain-dependent, as demonstrated by some differences between bacterial strains;

(iii) a large part of the antibacterial activity can be related to the high content of antioxidants in the wine;

(iv) overall, the winemaking process does not appear to exert a significant change on the antimicrobial properties of the wines analyzed, as demonstrated by comparable MIC values.

It is interesting to underline that Croatina Torchiato (CT), which is a wine characterized by a of lower economic value and therefore destined for a secondary market, turned out to be a very rich source of polyphenols. With chemical composition and antibacterial activity similar to those recorded in higher quality wines and, in view of its low cost, CT could be used as a source of novel anti-infective agents for the development of innovative biomaterials and coatings for medical devices and could be considered a valid alternative to other extracts obtained from tea, cranberry and cocoa.

Seen as a whole, our data indicate that polyphenols in red wines are capable of exerting a significant antibacterial action. Furthermore, the results of recent experimental studies show that polyphenols express

other influential activities / bioactivities, such as anti-inflammatory, antioxidant and cell membrane stabilizing effects. These results suggest that polyphenols could be advantageously and safely added to prosthetic biomaterials to counteract bacterial colonization, without resorting to the addition of toxic chemicals. Therefore, looking ahead, the incorporation of polyphenols into biomaterials can contribute to a new generation of delicate anti-infective materials, free from toxicity and with interesting biological properties.

The research that led to these results received funding from the project entitled "Experiences of Rice and Wine in the land of the Lombards and Visconti" approved by the Lombardy Region (Dds n. 11527—12 / 3/2014). The authors thank the vineyard "I Doria di Montalto" (MontaltoPavese, PV) and above all Daniele Manini for providing wine samples and providing us with technical support for the preparation of the Materials and Method Section.