Improved Properties and Microbiological Safety of Novel Cottage Cheese Containing Spices

Cott age cheese is a highly regarded dairy product. There has been increased interest in specialty cheese including cheese with additives like herbs, spices, or vegetables. The popularity of these cheese products is increasing due to their bett er biological value and improved fl avour. Herbs and spices are used in diff erent forms in food and traditional medicine because of their benefi cial impact on health (1,2). Spices contain phenolic compounds, one of the most important groups of natural antioxidants, which can reduce oxidative cell damage (3). In food production, phenols can reduce lipid oxidation and increase food stability (4). Numerous studies have reported antioxidant properties of many plants and spices (5– 11), but total phenolic content and polyphenol profi le of many of them are unknown due to the complex and varying chemical composition. Antimicrobial activity of herbal and spice extracts against diff erent types of microbes, including some foodborne pathogens have been investigated (4,12–15). Nevertheless, antibacterial activity of many spices is still unexplored, as well as their antimicrobial activity in real food system. ISSN 1330-9862 original scientifi c paper


Introduction
Cott age cheese is a highly regarded dairy product. There has been increased interest in specialty cheese including cheese with additives like herbs, spices, or vegetables. The popularity of these cheese products is increasing due to their bett er biological value and improved fl avour. Herbs and spices are used in diff erent forms in food and traditional medicine because of their benefi cial impact on health (1,2). Spices contain phenolic compounds, one of the most important groups of natural antioxidants, which can reduce oxidative cell damage (3). In food production, phenols can reduce lipid oxidation and increase food stability (4). Numerous studies have reported antioxidant properties of many plants and spices (5)(6)(7)(8)(9)(10)(11), but total phenolic content and polyphenol profi le of many of them are unknown due to the complex and varying chemical composition. Antimicrobial activity of herbal and spice extracts against diff erent types of microbes, including some foodborne pathogens have been investigated (4,(12)(13)(14)(15). Nevertheless, antibacterial activity of many spices is still unexplored, as well as their antimicrobial activity in real food system. Although there are many artisan and hand craft ed cheese, cream cheese, butt er or yogurt with vegetables on the market, to the best of our knowledge there are not any investigations of the eff ects of polyphenolic compounds on biological value of these milk products. Hayaloglu and Farkye (16) gave examples of specifi c European cheese with spices like Otlu cheese, Surk cheese, Kanterkaas and methods of their manufacture, but without reference to their biological value and microbiological safety. Prgica, Turoš or Kvargli are small, cone shaped, dried sour cheese with salt and dry red pepper, which are produced under diff erent names on the family farms and in small dairy plants in some parts of Croatia. Chemical composition and microbiological quality of Prgica and Turoš cheese have been investigated but data on the impact of red pepper on the biological value and shelf life of these cheese types were not given (17,18).
For this purpose, the major phenolic substances in fresh pepper and in some fresh herbs and corresponding dried spices as well as in the mixtures with chesse have been analysed and identifi ed. In addition, we report their antioxidant capacity and reducing power. Antibacterial activity of fresh pepper and fresh and dried herbs was determined in vitro and in situ.

Plant material and cheese
Dried spices, i.e. parsley, dill, pepper, garlic and rosemary were purchased at the store in the original packaging from diff erent origins and manufacturers: dill from Hungary and rosemary from Morocco (Kotányi GmbH, Obersdorf, Austria), pepper from Hungary (AGZ d.o.o., Zagreb, Croatia), and garlic from China and parsley from Hungary (Derma d.d., Varaždin, Croatia). During the summer, the fresh plants were collected from the areas of Slavonski Brod, Croatia. All the herbs were stored at 4 °C for a few days or were frozen at -20 °C before analysis. Fresh pasteurised cheese with less than 10 % fat in dry matt er served as a base for the addition of fresh pepper or fresh and dried herbs. A total of 30 combinations were made for testing sensory properties, since each plant (fresh or dried) was added at three diff erent mass fractions (0.5, 1 and 2 g per 100 g of cheese). Dill, parsley and rosemary were added in the form of chopped dried or fresh leaves. Cloves of garlic and sweet red horn pepper were cleaned and fi nely cut in a blender 800 ES (Snĳ ders Scientifi c, Tilburg, The Netherlands).

Preparation of plant extracts
Samples of fresh pepper and fresh and dried herbs were treated with 30 % ethanol or acetone. These solvents were selected with regard to the results achieved by Dragović-Uzelac et al. (19). Exactly 1 g of samples was weighed, extracted using 40 mL of 30 % aqueous ethanol (Merck, Darmstadt, Germany) or acetone (Merck) and homogenized. Extract mixtures were put in a water bath WNE 10 (Memmert, GmbH+Co. KG, Schwabach, Germany) at 60 °C for 60 min to refl ux. Aft er extraction, the content was fi ltrated in 50-mL volumetric fl asks and the volume was adjusted with 30 % aqueous ethanol or acetone up to the mark. Three replicates were done for each sample. The obtained extracts were used for spectrophotometric and high-performance liquid chromatography (HPLC) determinations as well as of antibacterial activity.

Total phenolic content
For the determination of total phenolic content (TPC), method with Folin-Ciocalteu reagent (Kemika, Zagreb, Croatia) was used (20). The absorbance was measured at 760 nm with the spectrophotometer DR 4000 U (Hach, Loveland, CO, USA). The results were calculated according to the calibration curve for gallic acid (Sigma-Aldrich, St. Louis, MO, USA), where y is absorbance at 760 nm and x is concentration of gallic acid in mg/L, R 2 =0.999. The TPC was expressed in mg of gallic acid equivalents (GAE) per 100 g of spices.
Results are presented as mean value of three replicates with standard deviation (S.D.).

Antioxidant activity assays
The antioxidant activity of the fresh pepper and fresh and dried herbs was determined by two diff erent methods: 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity assay and ferric reducing antioxidant power (FRAP) assay. DPPH method was based on the reduction of stable DPPH nitrogen radicals in the presence of antioxidants in the samples (21). DPPH values are expressed in mmol of Trolox equivalents (TE) per 100 g of the sample. On the other hand, the FRAP assay was conducted according to Benzie and Strain (22). This method is based on an increase of the absorbance at 593 nm due to the formation of tripyridyl-s-triazine complexes with Fe 2+ [TPTZ-Fe(II)] in the presence of a reductive agent from the samples. Results are expressed as mean value of three replicates±S.D.

High-performance liquid chromatography analyses of phenols
The HPLC system used was ProStar Varian equipped with a Varian Pro Star 330 photodiode array detector (Varian, Walnut Creek, CA, USA). The HPLC column was Nucleosil ® 5 μm C18 100A (Phenomenex, Torrance, CA, USA). The solvents for gradient elution were: A 0.2 % o--phosphoric acid, B methanol (J.T. Baker ® , Deventer, The Netherlands), C acetonitrile (J.T. Baker) in ultrapure water. The following gradient was used: 96 % A, 2 % B and 2 % C. The fl ow rate was 1.5 mL/min. Operating conditions were as follows: column temperature 30 °C and injection volume 20 μL. Chromatograms were recorded at 280 nm for phenolic acid and at 360 nm for fl avonoids. The identifi cation of the compounds was achieved by comparing the UV spectra and the retention times of the separated peaks with the retention times of the standards. Quantification was made by the external standard method using calibration of standards as a reference and was based on peak area from HPLC analyses and from mass fraction of compound. Calibration curves of the standards were made by diluting the stock solutions in 30 % aqueous ethanol. The mass fractions of phenolic acids were deter-mined according to the calibration curve of corresponding standards. Total fl avones were quantifi ed as apigenin and total fl avonols as quercetin.

Antibacterial activity of fresh and dried plants and their extracts in situ
In order to determine the antibacterial activity of the fresh and dried plants or their ethanol extracts in situ, pure culture of bacteria was incubated in nutrient broth, harvested, washed, resuspended in sterile water solution and added to cheese. Also, 0.5 to 2 g of fresh or dried plants or 2 mL of their ethanol extract (originally 0.04 g of spices) were added to 100 g of cheese and homogenized. The initial number of cells (given in Fig. 1 for each strain) and total viable cells aft er cheese storage (three days at 4 °C) were counted by standard dilution method on nutrient agar aft er incubation at 37 °C for 48 h. Results were expressed as mean value of three replicates±S.D.

Sensory analysis of cheese samples
Consumer sensory tests were conducted to evaluate the acceptability of the cheese with fresh pepper or fresh and dried herbs by the evaluation committ ee of twenty members, according to the method of Tratnik et al. (23). Taste, smell, texture and appearance were evaluated, where the taste was the most signifi cant att ribute. The maximum possible points were 20, and depending on the scores, cheese samples were grouped as follows: excellent (17.6 to 20.0), good (15.2 to 17.5), moderate (13.2 to 15.1), acceptable (11.2 to 13.1) and unacceptable (<11.2).

Phenolic content and antioxidant activity of extracts of fresh and dried plants and cheese samples
Total phenolic content and antioxidant activities measured by DPPH and FRAP assays in fi ve fresh domestic plants and dried commercial spices are shown in Table 1. Extracts of fresh plants in acetone had lower phenolic content except garlic and rosemary, while the extracts of dried plants in acetone had a slightly higher phenolic content than the ethanol extracts. Total phenolic content in fresh plants increased in the following order: red pep- per or garlic, followed by parsley, dill and rosemary. Phenolic content of dried plants was 2-4 times higher than of fresh plants, which is a consequence of the drying process and suggests that most of the antioxidants remain intact in the fi nal dried product. Among the tested plants, rosemary had the highest antiradical capacity, with the highest FRAP value of 17.1 to 26.4 mmol per 100 g, followed by dill, parsley, sweet red peppers and garlic. Total antioxidant capacity and reducing power are proportional to the content of phenolic compounds, which was confi rmed by high correlation coeffi cients between TPC and reducing power (data not shown).
High-performance liquid chromatography (HPLC) is widely used for both separation and quantifi cation of phenolic aglycones, whereas intact glycosides can be determined by HPLC with mass spectrometry. In this study, HPLC was used to quantify hydrolyzed aglycones in the samples of plants (Table 2). Gallic, p-coumaric, chlorogenic, caff eic and rosmarinic acids were monitored. Flavo-noids were expressed as total fl avones or total fl avonols since some peaks could not be identifi ed as aglycones. In parsley, gallic, p-coumaric and chlorogenic acids as well as apigenin and luteolin conjugates were detected. Flavonols and caff eic acid derivatives were found in dill samples, while aft er hydrolysis rosmarinic acid and caff eic acid conjugates were detected. The most abundant fl avonols were rutin and an unidentifi ed kaempherol derivative. Aft er hydrolysis, these peaks disappeared and those of quercetin and kaempherol aglycone increased. In fresh dill, phenolic acids were not detected probably because of the detection limit. The HPLC analysis of the ethanol extract obtained from the sweet red pepper pericarp showed that it contained low amount of polyphenols. Aft er acid hydrolysis, only luteolin, quercetin and a small amount of an unidentifi ed hydroxycinamic acid were detected. Low amount of polyphenols was detected in garlic, too. Rosemary extracts contained very high mass fractions of different phenolic compounds, among which rosmarinic acid was dominant. It was also found in dry dill (Table 2). The results are expressed as mean value±S.D. GAE=gallic acid equivalents, TE=Trolox equivalents The results in Table 3 show that the biological value of cheese can be improved by adding the plants rich in bioactive phenolic compounds. Cheese samples with added fresh or dried plants had higher TPC and free radical scavenging activity than the control sample. Selection of samples for further processing was based on the sensory analysis. Among diff erent cheese samples, the cheese with dill had the highest level of TPC (37.82 mg of GAE per 100 g), on a par with rosemary (34.54 mg of GAE per 100 g) due to the highest level of phenolic compounds in these spices. Cheese with rosemary had strong antioxidant activity.
The total phenolic contents of cheese samples were measured with Folin-Ciocalteu reagents, which do not measure only phenols, and can react with many substances from the cheese. Also, a number of factors aff ect the interactions between phenolic molecules and protein interactions (pH, temperature, phenolic structure, molecular mass and amino acid composition). This can explain the diff erences in phenolic content of plants and their contribution to antioxidant activity of cheese.

In vitro and in situ antibacterial properties of plant extracts
Antibacterial activity of the ethanolic extracts of plants against some foodborne pathogens, Gram-positive and Gram-negative bacteria, is shown in Table 4. These bacterial species are the most common contaminants of traditional homemade cheese and cream (24). Plant extracts inhibited the growth of most bacterial isolates with the wide inhibition zone in the range from 9.  The results are expressed as mean value±S.D.
tance to the extracts of fresh parsley, dill, garlic and sweet red pepper probably due to the lower mass fraction of phenolic compounds in these plants. However, these extracts showed signifi cant antibacterial activity against Escherichia coli. Extracts of dried plants inhibited the growth of more or less all tested microorganisms. Among the extracts, rosemary showed very promising and significant antibacterial activity, especially against Gram-positive bacteria (Table 4). Only dill and rosemary reduced Listeria monocytogenes growth. Signifi cantly diff erent results were achieved in the real system when the antimicrobial activity of plant extracts and fresh plants in cheese samples was tested. Although the number of pathogens was lower in the cheese with added supplements, statistically signifi cant diff erence was recorded only in the cheese supplemented with spice extracts. Rosemary extract notably inhibited bacterial growth, except L. monocytogenes (Fig. 1).

Sensory scores of cheese samples
Cheese samples with the addition of 0.5, 1 and 2 g of fresh or dried plants per 100 g were selected for sensory analysis (Fig. 2). Acceptance of 30 cheese samples on the basis of overall liking by a consumer panel was conducted. The results showed bett er acceptability of cheese with fresh pepper or herbs in relation to the dry spices.

Discussion
In this paper we investigated the possibility of the use of fresh or dried pepper and herbs as cost-eff ective natural antioxidants and food preservatives in the production of cott age cheese with extended shelf life, accept-able sensory properties and benefi cial antioxidant capacity. Herbs and spices possess essential, aromatic oils, which show antimicrobial activity and contribute to the aroma and taste of food, as well as phenolic compounds, one of the most important groups of natural antioxidants. Some researchers have already described the potential of milk or cheese supplementation with polyphenols with the aim to develop novel milk products with incorporated nutraceuticals and bioactive compounds. Cinnamon, oregano, clove, pomegranate peel, grape, green tea extract, dehydrated cranberry powder, cardamom and nutmeg were added as functional ingredients during the cheese or yogurt making process (4,(25)(26)(27)(28). Except these, common herbs and spices added to cheese include green peppers, black peppercorns, horseradish, thyme, cumin, caraway, tarragon, basil, onion, as well as sun-dried tomatoes, depending on the country and tradition (16). Some spice extracts like oregano and clove can eff ectively inhibit lipid oxidation in cheese during storage and thus help to preserve the cheese sensory characteristics (4). Adding herbs or spices must not aff ect the metabolism and activities of the applied starter culture during fermentation and ripening. The addition of plants to the cott age cheese aft er cheese making process annulled this problem and it did not have an impact on the physical and chemical characteristics of the cheese in our research (protein and fat content, dry matt er, ash and pH; data not shown). Parsley, dill, pepper, garlic and rosemary were investigated because these plants are commonly consumed.
Dill and parsley belong to the same Apiaceae family, and their TPC values were in close proximity. Rosemary belongs to botanical family Labiatae, also called Lamiaceae, comprising many widely used culinary herbs, such as oregano, basil, mint, sage or marjoram, all rich in phenolic compounds (25,(29)(30)(31). The literature data about total phenolic content of spices is very diffi cult to compare since it can be infl uenced by chemical structure, the extraction method employed, sample particle size, storage time and conditions, as well as presence of interfering substances. However, the results of Alezandro et al. (1) and Santos et al. (31) are similar to those obtained here, confi rming the high TPC and antioxidant activity of the alcoholic extract of rosemary. The literature data about 18 Chlorogenic acid was the most abundant phenolic acid detected in parsley, diff erent from Kaiser et al. (36), who detected p-coumaric acid derivatives as the most abundant class of phenolics. Flavones are present in signifi cant concentrations and apigenin-7-O-apiosylglucoside (apiin) is the major compound in parsley (32,36). Phenolic compounds are found in dill fl ower, leaf and seed but the fl ower extract had the highest total amount of polyphenols (37). Chlorogenic, p-coumaric, gallic, benzoic and p-anisic acids are the major phenolic acids detected in dill fl ower extract, while myricetin, quercetin, luteolin and kaempferol are the most abundant fl avonoids (37). Chlorogenic, caff eic and rosmarinic acids, quercetin and kaempferol, as well as high antioxidant activity were determined, probably due to the presence of quercetin and rosmarinic acid. Quercetin has a hydroxyl group at C-3 position in the ring which results in greater free radical scavenging effi ciency (38). Rosmarinic acid has two ortho-dihydroxy groups, which is the most important structural feature for strong antioxidant activity (38). Rosemary, containing rosmarinic acid, had the highest antioxidant activity. Consequently, cheese with rosemary had strong antioxidant activity. Phenolic diterpenes, rosmarinic acid and hydroxycinnamic acid ester are the major antioxidant compounds existing in rosemary (39). In peppers and garlic, low amounts of polyphenols were detected. Nevertheless, total antioxidant capacity and reducing power of peppers were high due to the presence of other health-related compounds such as ascorbic acid, carotenoids, tocopherols and capsaicinoids (40). Bae et al. (41) extracted quercetin, luteolin, kaempferol and myricetinin in descending order from ripe peppers with diff erent extraction solvents. There are only a few research papers about the phenolic composition of garlic (42,43). Beato et al. (43) determined total phenolic content in garlic to be in the range from 3.4 to 10.8 mg of GAE per g of dry mass and caff eic and ferulic acids were the major phenolic acids.
Food safety is a fundamental concern for both consumers and food producers. In addition to the antioxidant activity, spices have a strong antibacterial activity and can be used as food preservatives (12,14,15,44). Shan et al. (12) have suggested that the antibacterial activity of spices and herbs extracts is associated with their phenolic constituents and essential oils. Our results do not show close correlation between the mass fraction of phenolic compounds and the extract inhibition zones probably due to the presence of other compounds with antibacterial activity. Fresh and dry plants showed a variable antibacterial activity against the tested strains despite the higher mass fraction of phenolics in dry spices. Garlic and peppers have a low content of polyphenols, but signifi cant antibacterial activity was observed. Allicin, an organosulphur compound present in garlic, acts as a growth inhibi-tor of both Gram-positive and Gram-negative bacteria (45,46). Lanzott i et al. (47) have reported that a bactericidal property of garlic-derived organosulphur compounds was much greater compared to phenolic compounds. Capsaicinoid, a naturally occurring alkaloid group in peppers, contributes to the pungency, taste and aroma of peppers and acts as the main antibacterial component in pepper (48). Rosemary showed the strongest antimicrobial activity, especially towards the Gram-positive bacteria. Gram-positive bacteria were more sensitive to rosemary extract and other plant extracts than Gram-negative ones due to the diff erence in the structure of cell membrane (49). Only dill and rosemary reduced L. monocytogenes growth probably due to the presence of rosmarinic acid in these spices. Moreno et al. (50) concluded that carnosic and rosmarinic acids are the main antimicrobial compounds present in rosemary extracts. Phenolic compounds containing hydroxyl group are more eff ective against microorganisms compared to those that contain the carbonyl group. The -OH group can easily bind the active site of enzymes, altering cell metabolism of microorganisms. Additionally, the position of the -OH group in the phenolic ring structure infl uences antibacterial and antioxidant activities (38).
There is litt le information about the antimicrobial activity of spices used in real food systems. The eff ect of spices on food pathogens depends on their phenolic content and interaction with various food components. Zaika (51) summarised the factors that can aff ect the antimicrobial activity of herbs and spices. The medium conditions like pH, NaCl content and temperature infl uenced the antibacterial activity of the rosemary extract and can have a synergistic eff ect with the extract of spices (29). Lipids, surface-active agents, and some proteins can infl uence the antibacterial activity of spices. Because of that, a large number of results that confi rmed the antimicrobial activity of plants achieved in vitro must be verifi ed in real media. Our study did not confi rm all antimicrobial activity results achieved in vitro. Rosemary extract in the real system did not inhibit the L. monocytogenes growth. L. monocytogenes is more tolerant than most foodborne pathogens over a wide range of environmental conditions (52), among others, due to resistance to low pH, which was in cheese samples pH=4. 28-4.31. On the other hand, some authors explained that the amounts of spices and herbs added to food for fl avour are generally too low to prevent spoilage by microorganisms (53). Our results refute this view and confi rm that phenolic compounds, among other compounds, are responsible for the antimicrobial activity of plants. Namely, ethanolic extract of phenols from only 0.04 g of plants effi ciently reduced bacterial growth (Fig.  1), contrary to the ten times higher mass fraction of fresh plants (data not shown). We assume that the extraction of phenolic compounds was not suffi cient to reduce the bacterial growth during cheese production. The extraction effi ciency of phenolic compounds from plant material greatly depends on the solvent (Table 1) and water is less eff ective than ethanol, methanol and acetone or their aqueous mixtures (19). Also, it is generally supposed that the high levels of fat and/or protein in foodstuff s (cheese contained 10.86-11.81 % of proteins and 8.33-9.75 % of lipids on dry mass basis) can protect the bacteria from the activity of the phenols and essential oils. In the lipid phase, the solubility of hydrophobic compounds increases, and they are less available to act on bacteria present in the aqueous phase (54).

Conclusion
Recent trends in food industry and thus in cheese manufacture focus on the production of novel functional products with health benefi ts. The results show that cheese samples with fresh pepper and fresh or dried herbs are naturally fl avoured products with acceptable sensory properties, highly nutritive value and good microbiological quality, which can be produced in short time. Fresh plants are more acceptable by consumers, but dry ones contribute more to the biological value. The antibacterial activity and antioxidant activity of plants originates from their phenolic compounds, which are considered as powerful bioactive compounds expressing strong antibacterial and antioxidant activities. The fresh and dried plants mixed with cheese to add fl avour could aid in prolonging storage at refrigeration temperatures, at which the multiplication of microorganisms is slow. Phenolic extracts from plants have a potential as natural preservatives and antioxidants.