Anmicrobial Activity and Composition of Different Cultivars of Honeysuckle Berry Lonicera caerulea L. †

The aim of this work was comparative study of composition and antimicrobial properties in eleven cultivars of honeysuckle berries. Using spectrophotometric methods, we compare the content of total phenolic compounds (TPC) and anthocyanis, chromatic characteristics of berries, which were grown in collection of Vytautas Magnus University Botanical Garden and collected at maturation stage. Also, content of ascorbic acid and saccharides were evaluated by HPLC using diode ray and light scattering detectors. Antimicrobial activity of ethanolic and water extracts of honeysuckle berry was evaluated by the agar well diffusion method. Bacterial tests have identified antimicrobial properties of honeysuckle berries against undesirable in food products bacteria but without affecting Candida and Saccharomyces cerevisiae yeast. The cultivar ‘Morena’ had the highest anthocyanins (781 mg/100 g) and total phenolic compounds (799 mg/100 g), the lowest anthocyanins (282 mg/100 g) and TPC (300 mg/100 g) content was detected in ‘Vostorg’ cultivar. Cultivars ‘Pavlovskaja’ and ‘Pereselenka’ had high content of ascorbic acid. The maximum glucose and fructose content were detected in ‘Leningradskaja’ cultivar.


Introduction
For healthy diet it is recommended to consume at least 400 g of vegetables and fruit per day. Also according World Health Organisation recommendations consumption of sugar should be lowered and increased amount of dietary fiber. Berries are good source of dietary fibre, polyphenols, ascorbic acid and other bioactive compounds [1]. The edible blue honeysuckle comes from Russia and in the recent years has been considerably planted in some European countries, Lithuania is among them. Its interesting characteristics are high resistance to cold, different soil acidities, pests and various diseases [2]. The berries are rich in an ascorbic acid and phenolic contents, which have nutritional and health promoting properties for humans. In Japan honeysuckle berries are used in traditional medicine for slowing the aging process, preventing heart diseases and gastrointestinal dysfunction [3]. Anthoyanins, plant pigments, responsible for red to blue colour of fruits, are the biggest contributors to total phenolics in blue honeysuckle berries. High content of cyanidin-3glucoside suggest their good antioxidant, anti-inflammatory, antimicrobial, cardioprotective and hepatoprotective activities [4,5]. In recent years increased consumer demand for the use of plant extracts as natural antimicrobial agents in food instead synthetic food additives. Some studies have shown antimicrobial properties of honeysuckle berries. Phenolic acids present in honeysuckle berries can act as natural antimicrobial agents to control Candida parapsilosis, Enterococcus faecalis, Escherichia coli, Staphylococcus epidermidis, or Streptococcus mutant [6]. Antibacterial effects of ethanol and buffered water infusions obtained from freeze-dried fruits of L. caerulea were studied on gram-positive bacteria (Listeria monocytogenes, Kocuria rhizophila, Bacillus subtilis), gram-negative foodborne pathogenic bacteria (Escherichia coli, Campylobacter jejuni) and gram-positive bacteria selected as probiotic bacterial species often used in dairy products (Bifidobacterium bifidum, Lactobacillus acidophilus). It has been found that some blue honeysuckle infusions can elicit high antimicrobial activity against foodborne pathogens without strong inhibition towards probiotics. Consequently, tested probiotic bacteria with honeyberry extract can be used in the food processing industry as potential antimicrobials and functional components [7].
The studies on bioactivity of honeysuckle berries started only recently, the data are limit. It is well known that composition of bioactive compounds depends on genotype, climatic conditions, agronomic practices. This is preliminary report on honeysuckle berries planted in Lithuania. The aim of this study was comparative study of composition and antimicrobial properties in eleven cultivars of honeysuckle berries grown in Lithuania.
Antimicrobial activity was evaluated by the agar well diffusion method. Different extracts were prepared from homogenized berries to analysis. 2 g of berry paste were extracted with 8 mL of water in an ultrasonic bath (Ultrasonix cleaner proclean 3.ODSP) for 30 min. It was then centrifuged for 10 min at 5300 rpm (Labofuge 200 Heraeus Thermo Scientific. Rotor 3760, 2700 g). Ethanolic extract was prepared from 3 g of berry cakes and 10 mL of ethanol. Extraction and centrifugation conditions were the same. Obtained supernatants were used for evaluation of antimicrobial activity. Undesirable in food products the yeasts and bacteria were used in the test cultures. Bacteria were grown in peptonesoy bouillon. Yeasts were grown on a slant potato dextrose agar. Eight-millimeter diameter wells were pushed in the agar and filled with 50 μL of sample. The plates were incubated overnight at 37 °C. After incubation the inhibition zones were measured and the effect was calculated as a mean of three replicate tests.

pH, Soluble Solids and Dry Matter Amount of Honeysuckle Berries
Eleven cultivars of Lonicera caerulea L. were investigated in this study. Berries were grown in the Vytautas Magnus University Botanical Garden collection. Only fruits at maturity stage, based on the colour and texture were collected. After crushing the berries pH, total soluble solids and dry matter amount was evaluated (Table 1).

Ascorbic Acid Content
Another important parameter in the composition of berries is the amount of ascorbic acid. It was in average 3.3 mg/g DM. Unusually large amnount of vitamin C was found in 'Pereselenka' (10.5 mg/g DM) and 'Pavlovskaja' (7.6 mg/g DM) cultivars ( Figure 2). If calculate the average value after excluding maximum values it would be 34.3 g/100 g and 2.28 mg/g DM. Vitamin C content was the lowest in 'Nimfa' cultivar-13.46 g/100 g and 0.73 g/g DM. Ascorbic acid content reported for honeysuckle beries grown in Switzeland were 1.78-4.21 mg/g DM [1], grown in Slovenia-17.75-25.77 mg/100 g [2]. The differences coud be result of different extraction techniques and growing conditions.

Chromatic Properties of Honeysuckle Berries
Cromatic properties of honeysuckle berries were presented in Table 2. The L* values of the Lonicera caerulea L. cultivars were not variable and ranged from 23.34 to 26.44. The cultivar 'Pereselenka' was characterized by a high a* (13.15) and b* (5.33) values, also the chroma C (14.18) and hue angle h° (0.38) for this cultivar was the highes. The lowest values of C and h° were determined in 'Balalaika' cultivar, 6.90 and 0.18 respectively. 24.41 ± 0.16 6.79 ± 0.04 1.23 ± 0.06 6.90 ± 0.03 0.18 ± 0.01

Total Phenolic Compounds and Anthocyanins Content
Total phenolic compounds and anthocyanins content presented in Figure 3. The average content of total phenolic compounds expressed in mg of gallic acid equivalents was 492.1 mg/100 g and 31.6 mg/g DM, total anthocyanins-451.5 mg of cyanidin-3-glucoside equivalents/100 g and 29.2 mg of cyanidin-3-glucoside equivalents /g DM. Literature refers to values between 8.4 and 65 mg cyanidin-3-glucoside equivalents/g DM and from 7.0 to 57.1 mg gallic acid equivalents/g DM [1]. The cultivar 'Morena' had the highest anthocyanins (781 mg/100 g) and total phenolic compounds (799 mg/100 g), the lowest anthocyanins (282 mg/100 g) and TPC (300 mg/100 g) content was detected in 'Vostorg' cultivar. The same tendency was observed after conversion to mg/g DM.

Antimicrobial Activity
Investigation of antimicrobial activity showed that aqueous extracts of honeysuckle berries weakly inhibit the growth of gram-negative and gram-positive test cultures (Table 3).
B. subtillis were the most sensitive, zones of inhibition were 9.5-11.0 mm. S. typhimurium showed largest resistance to aqueous berry extracts, only 'Kalinka' and 'Balalaika' cultivars showed 9.0 mm and 8.5 mm zones of inhibition. No inhibition zone was observed against yeast C. albicans and S. cerevisea.
Berry cakes, by-product of juice extraction, are of particular interest because they could be used as a cheap raw material for functional ingredients production. Investigation of antimicrobial activity of ethanolic extracts of berry cakes showed greater inhibition effect compare to aqueous berry extracts on test cultures (  Table 3. The antimicrobial influence of aqueous berry extracts on test cultures. Inhibition zone in mm, including the 8 mm hole. If inhibition zone wasn't observed, results is presented as 0.

Conclusions
Investigation of the antimicrobial properties showed that European cranberry extracts inhibited the growth of wide range of human pathogenic bacteria, both gram-negative (Escherichia coli and Salmonella typhimurium) and gram-positive (Enterococcus faecalis, Listeria monocytogenes, Staphylococcus aureus, and Bacillus subtilis).
Author Contributions: I.J. conceived and designed the experiments, performed HPLC analysis, analyzed the data, and wrote the paper. A.P. performed preparation of sample, spectrophotometric asay and other measurements. A.Š. performed antimicrobial activity experiment, analyzed the data. L.Č. provided berry material and also did a great contribution in preparing the manuscript. All authors have read and agreed to the published version of the manuscript.

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

Abbreviations
The following abbreviations are used in this manuscript: Publisher's Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.