Dear Label_Name,

Greetings! I hope you are well.

I am writing on behalf of an international publishing house, Science and Education Publishing, USA (http://www.sciepub.com).

Science and Education Publishing (SciEP) is a multidisciplinary open access publisher of Journals covering the fields of Science, Technology and Medicine. All SciEP publications are subject to high-quality peer review, editorial and production processes and are freely available online.

Welcome to the April, 2013 edition of SciEP newsletter, providing monthly summaries of articles on science & education related topics of interest to scientist and professionals. The mission of our journals is to provide an open access forum for publishing new findings on issues pertinent to scientific and academic field. We also invite you to submit original research papers, review articles, short reports, or case studies are considered for publication in our Multidisciplinary Journals.

American Journal of Public Health Research
DOI: 10.12691/ajphr-1-1-3

The Implementation of a Tele-Homecare System with Service Oriented Architecture and HL7 Message Transmission Standard
Ching-Sung Wang

American Journal of Medical and Biological Research
DOI: 10.12691/ajmbr-1-1-2

The Influence of Ethanol on Pyruvate Kinases Activity in Vivo, in Vitro, in Silico
Lelevich Sergey Vladimirovich, Khrustalev Vladislav Victorovich, Barkovsky Eugene Victorovich, Shedogubova Tatyana Aleksandrovna

American Journal of Medical Sciences and Medicine
DOI: 10.12691/ajmsm-1-1-1

Clinical and Histological Features in Wegener Granulomatosis
Li Cavoli Gioacchino, Passantino Rita, Bono Luisa, Tortorici Calogera, Ferrantelli Angelo, Giammarresi Carlo, Rotolo Ugo

American Journal of Infectious Diseases and Microbiology
DOI: 10.12691/ajidm-1-1-2

The Curse of Lymphatic Filariasis: Would the Continual Use of Diethylcarbamazine Eliminate this Scourge in Papua New Guinea?
Anne Lanham, Lillian Mwanri

We have released issues of some journals on February 2013.
American Journal of Public Health Research
American Journal of Medical Sciences and Medicine
American Journal of Medical and Biological Research
American Journal of Pharmacological Sciences
American Journal of Clinical Medicine Research
American Journal of Nursing Research
American Journal of Cancer Prevention
American Journal of Medicine Studies
American Journal of Infectious Diseases and Microbiology
American Journal of Cardiovascular Disease Research
American Journal of Biomedical Research
American Journal of Mechanical Engineering
American Journal of Microbiological Research
Journal of Preventive Medicine
American Journal of Food and Nutrition
American Journal of Hypertension Research
American Journal of Sports Science and Medicine
Journal of Cancer Research and Treatment
American Journal of Zoological Research
Journal of Biomedical Engineering and Technology
American Journal of Microbiological Research
Biomedical Science and Engineering

We are pleased to announce that submissions are now being accepted for peer review for all the journals across all our subject areas in SciEP.

Please submit your manuscript via our online submission system:
http://www.sciepub.com/author/signin

To unsubscribe from our mailing list, please click on the following link: unsubscribe

I am looking forward to hearing from you.

Kind regards,

David Rain
Email:join@sciepub.com
Science and Education Publishing (http://www.sciepub.com)

Analysis of the Flavor and Active Compounds in Lonicera japonica Wine Produced by a New Extraction and Fermentation Method

Xiaolong Zhou, Orlando Borras-Hidalgo, Wenting Ruan, Xinli Liu*, Guoxiang Lin

State Key Laboratory of Biobased Material and Green Papermaking, Qilu University of Technology, Shandong Academy of Sciences, Jinan, China

Abstract: Lonicera japonica species have been used as an adjuvant therapy for physical fitness, due to their extensive biological activity and pharmaceutical properties. For example, this species demonstrates antimicrobial, anti-inflammatory and detoxifying effects. Therefore, the changes in the chlorogenic acid, amino acid, and total polyphenol contents and in the antioxidant activity were analyzed. The results showed that the content of chlorogenic acid was 2378.63 mg/L without residue fermentation, 2039.05 mg/L with residue fermentation and 1476.74 mg/L with liquor extraction, which were increases of 1.86, 1.53 and 0.87 times more, respectively, than the chlorogenic content in unfermented L. japonica broth. The total content of polyphenols during the fermentation time had a positive linear correlation and the following values were obtained; fermentation without residue was 0.73 mg/mL, fermentation with residue was 0.62 mg/mL, and liquor extraction was 0.45 mg/mL. The clearance rate of ABTS and DPPH reached approximately 95%. Moreover, flowers could be used to avoid the production of methanol and fusel oil. Therefore, the nutrients and active functional components in L. japonica and the flower flavor of L. japonica could be preserved. This suggested fermentation process is better than the liquor extraction process and is a safe and controllable metabolic process.

Keywords: Lonicera japonica, chlorogenic acid, polyphenol, antioxidant activity, liquid fermentation

Received August 11, 2020; Revised September 14, 2020; Accepted September 29, 2020

Citation: Xiaolong Zhou, Orlando Borras-Hidalgo, Wenting Ruan, Xinli Liu, Guoxiang Lin, Analysis of the Flavor and Active Compounds in Lonicera japonica Wine Produced by a New Extraction and Fermentation Method. American Journal of Food Science and Technology, 8 (5). 185-195. January 2020. doi:10.12691/ajfst-8-5-3

1. Introduction

L. japonica is a species of the Lonicera genera of dry buds and primary flowers, which are also known as honeysuckle in China, and commonly used in traditional Chinese herbal medicine [1] to treat pneumonia, swelling, infection, and breast cancer [2,3]. This species has important potential uses in pharmaceutical preparations, cosmetics and health care products, such as toothpastes, herbal teas and food additives in beverages [4,5]. This species produces various natural bioactive compounds with effects on human health, such as antiviral and anti-inflammatory reactions [6,7,8] Additionally, these bioactive compounds can significantly enhance the immunological function of human cells and body fluids, exhibiting a high effect in aqueous solution. Previous studies have shown that L. japonica has a significant inhibitory effect on Staphylococcus aureus and Streptococcus pneumonia [9]. Additionally, L. japonica extracts have anticoagulation, hypolipidemic and hypoglycemic effects [10]. These pharmacological effects are derived from the various functional active substances, such as chlorogenic acid, volatile oil, flavonoids, pectin [11], saponins, inositol, luteolin, polysaccharides, phenolic acids and iridoids [12,13].

In particular, the most important active ingredient is chlorogenic acid, which is an ester compound formed by the condensation of quinic acid (QA) with trans-cinnamic acid (t-CA) [14]. A previous study revealed for the first time the structural formula of chlorogenic acid. Common trans-cinnamic acids are caffeic acid, p-co-phthalic acid (p-CoA) and ferulic acid [15]. The type and amount of hydride affects the esterification of trans-cinnamic acid with quinic acid. Thus, chlorogenic acid has a variety of isomeric compounds, the most common of which is 3-O-caffeoylquinic acid (3-CQA), which is commonly known as chlorogenic acid [16].

At present, the biological function of chlorogenic acid has been widely used in the food, beauty, medicine and chemical industries [17] because chlorogenic acid has the following wide range of benefits for the treatment of clinical diseases: antioxidant, analgesic, and antibacterial behavior [18]; liver and nerve protection [19]; heat removal; detoxification; antiradiation, anti-obesity, and antihypertensive behavior [20,21]; immune system regulation and nerve center activation [22,23]. In addition, studies have shown that chlorogenic acid can regulate lipid metabolism and the physiological metabolism of glucose as well as diseases related to a healthy metabolism, thereby assisting in the treatment of many diseases, such as liver steatosis, cardiovascular disease, diabetes and obesity [24].

L. japonica wine is a type of deeply processed product with flowers as raw materials. People commonly drink L. japonica tea or other L. japonica products to gain certain effects. The idea of brewing L. japonica wine is based on an ancient Chinese pharmacopeia that included some flower-soaked wines that could cure some diseases according to the characteristics of the active ingredients in different flowers [25]. However, the active ingredients in these wines are only obtained through a simple extraction method, and the pharmacological effects have been poorly studied. The development of a new type of L. japonica wine and its effects constitute a new focus on the production of low-grade wine with health care functions. In this study, we aimed to compare two methods of fermentation and extraction and their effect on the bioactive components in L. japonica wine, thus providing a basis for the development of wine with health benefits.

2. Materials and Methods

2.1. Chemicals and Reagents

Chlorogenic acid and amino acid standards (purity 98.0%, HPLC grade) were obtained from Shanghai Yuanye Bio-Technology Co., Ltd. (China) and Alta Technology Co., Ltd (USA). Formic acid was obtained from Merck (Germany). Other analytically pure reagents used in this study were from Sinopharm Group Chemical Reagent Co., Ltd (Shanghai, China).

2.2. Microorganisms and Culture Conditions

Saccharomyces cerevisiaeEC1118 was obtained from Presque Isle Wine Cellars (Pennsylvania, USA). The use of this yeast is advantageous due to its good comprehensive fermentation ability, while having the best tolerance for high alcohol concentrations that are between 18 and 20%. Furthermore, EC1118 yeast can ferment a variety of vegetables and flowers but not fruits.

Aroma-producing yeast was obtained from Angel Yeast Co., Ltd., Hubei, China. Additionally, yeast can enhance wine flavor.

Saccharomyces cerevisiae EC1118 strain activation: Dry yeast (1 g) was dissolved in 9 mL of a sterile saline solution 100 times, and then 100 µL of the yeast solution was absorbed and coated on a solid plate of yeast extract/peptone/dextrose (YPD) medium and placed in an incubator at 28°C for 48 h. Saccharomyces cerevisiae with a full colony shape was selected and inoculated in wort medium (wort 70 mL, natural pH of 6.4, the plate was inverted and sterilized at a high temperature for 20 min) for activation and cultured in a shaking table at 28°C and 150 r/min for 16 h to obtain a seed liquid. The seed liquid inoculation scale was 5% (v/v), which was then joined with a new wort medium to continue culturing according to the culture method, thereby obtaining the yeast activation solution.

Aroma-producing yeast activation: A 2.5% saccharose solution (mass fraction) was mixed with 10 times the amount of dry yeast, and the temperature was adjusted to 35°C. The yeast was dissolved in the saccharose solution and activated at 35°C for approximately 1 h. In the process of fermentation, the inoculation scale was 0.1% (v/v).

2.3. Preliminary Treatment of Raw Materials and Fermentation Pathways of L. japonica

The optimum time to harvest L. japonica is early June in the morning, when the buds are not completely opened, remain full of nutrients, and have a strong smell and good color. Harvested buds were vacuum dried at 50°C and 0.09 MPa for 16 h.

2.4. Dry Processing

First, L. japonica was stored at -80°C and dried at 50°C and 0.09 MPa for 16 h in a vacuum drying oven according to the vacuum drying method [26]. This process was used to obtain dried L. japonica. After treatment, the water content of L. japonica was less than 5%, so its fragrance was completely released.

2.5. Preparation of Fermentation Broth

L. japonica broth was divided into three batches.

The first was fermented without residue. L. japonica (50 g) and pure water (200 mL) were mixed and crushed in a beater for 2 min at 60000 r/min. The samples were filtered through gauze to obtain a clarified broth.

The second was fermented with residue. L. japonica (50 g) and pure water (200 mL) were mixed and crushed in a beater for 2 min at 60000 r/min. It was not filtered in the process and contained residue.

Liquor extraction was kept as the control group. L. japonica (50 g) and 200 mL anhydrous liquor were mixed and crushed in a beater for 2 min at 60000 r/min.

These three batches of L. japonica broth were poured into consecutive fermentation tanks, and 800 g of pure water was added. Each treatment had three replicates.

2.6. Fermentation Process

The addition of 150 g glucose was dissolved fully, and the food grade citric acid was adjusted to pH=3.5-4. To dissolve the L. japonica plant and release the fermentable sugars, 1 g pectinase and 1.5 g cellulase were added to hydrolyze L. japonica at 40°C for 30 min. After that, the broth was sterilized at 115°C for 20 min, inoculated with Saccharomyces cerevisiae EC1118 (0.3% v/v) and aroma-producing yeast (0.1% v/v), and then shaken well at a rotation speed of 150 r/min for 12 h. Alcohol fermentation was carried out under aseptic conditions for 30 days at 25°C. The fermentation process was performed according to referenced methods [27,28,29].

Finally, L. japonica wine was obtained by fermentation and extraction (Figure 1).

Figure 1. Brewing process of Lonicera japonica wine

2.7. Component Detection

Qualitative analysis of flavor compounds by GC-MS in L. japonica wine

Chromatographic conditions: Inlet temperature of 220°C, column temperature of 35°C for 5 min, ramp rate of 5°C/min to 100°C for 2 min, ramp rate of 15°C/min to 230°C for 10 min, carrier gas flow rate of 1 mL/min, and column db-wax (60 m×250 µm×0.25 µm).

Mass spectrometry conditions: The 70 eV electron ionization (EI) mode of the EI ion source at 230°C and a scanning mass range of 30-500 amu.

After centrifugation, the sample passed through a 0.22 µm filter membrane, and then 5 mL was injected into an analysis bottle for machine testing.

Sample processing: The fermentation and extraction broth was directly taken and centrifuged for 5 min at 5000 r/min. Then, approximately 1 mL of supernatant was decanted, passed through a 0.45 µm filter membrane, and injected into the sample bottle with a syringe. Approximately 2 mL of the wine sample was placed into a 15 mL headspace bottle, and 5 mL of deionized water was added, saturated with NaCl, and extracted with ultrasonication for 30 min.

Injection sample method: Headspace solid phase microextraction.

2.8. Quantitative Analysis

Determination of methanol, ethanol, total acid, pH value, and ethyl acetate in Lonicera japonica wine [30].

The glucose content was measured by using the SBA-90 biosensor.

2.9. Determination of the Total Polyphenol Content

The total polyphenol content of L. japonica wine was determined using the Folin-Ciocalteu method [31].

Preparation of the standard curve: The standard sample was diluted 0, 10, 50, and 100 times. Additionally, 5.0 mL of 10% Folin reagent was added, and the reaction was shaken for 5 min. Then, 4.0 mL of sodium carbonate solution (7.5%) was added and shaken to blend the constituents together. After reacting for 60 min at room temperature, the absorbance was measured at 765 nm. Finally, according to the experimental OD value to confirm the optimal dilution, a standard curve of polyphenols was established. Additionally, the fermentation broth was determined using the method described above.

2.10. Detection of Chlorogenic Acid

The content of chlorogenic acid was determined according to the national standard detection method [32]. A high-performance liquid chromatography (HPLC-20A, SHIMADZU) instrument was used for detection.

Liquid chromatography conditions: ODS C18 column (250 mm×4.6 mm, 5 μm), flow rate of 1.0 ml/min, column temperature of 35°C, sample volume of 10 μL, detection wavelength of 325 nm, and a 9:1 ratio of 0.5% acetic acid solution:acetonitrile. The standard curve of chlorogenic acid was established based on the peak area, and according to the standard curve, the content of chlorogenic acid in the L. japonica wine samples was calculated.

2.11. Analysis of the Antioxidant Activities of Lonicera japonica Wine

ABTS (2,2'-diazo-bis-3-ethylbenzothiazolin-6-sulfonic acid): The ABTS radical scavenging activity was determined using a modified method [33]. The prepared working fluid (ABTS+K₂S₂O₈) was diluted 40 times with 95% ethanol (analytical grade) so that the absorbance was 0.7±0.02 at 734 nm.

ABTS reserve solution (7.4 mmol/L): A mixture of 96 mg of ABTS and 25 mL of distilled water.

K₂S₂O₈ reserve solution (2.6 mmol/L): Briefly, 378.4 mg of K2S2O8 and 10 mL of distilled water were mixed together. Then, 5 ml of 7.4 mmol/L ABTS reserve fluid and 88 µL of 2.6 mmol/L K₂S₂O₈ were mixed and allowed to stand for 12-16 h to produce the ABTS working fluid.

Sample experiment: Briefly, 3.9 mL of the diluted solution was mixed with 0.1 mL of sample, and the absorbance was measured at 734 nm after standing at room temperature for 6 min in the dark. The result was recorded as the absorbance of sample A. Then, 3.9 mL of the dilution was mixed with 0.1 mL of 95% ethanol solution for 6 min, and the absorbance was measured at 734 nm. As a result, A₀ was recorded as a blank control group and A was recorded as a sample result.

DPPH (1,1-diphenyl-2-trinitrophenylhydrazine): The DPPH radical scavenging activity was determined using a modified method [34]. In brief, 7.8 mg of DPPH was weighed in a volumetric flask, dissolved in anhydrous ethanol and diluted to the desired volume in the dark. Then, 3 mL of DPPH-ethanol solution was added to 3 mL of sample solution A. It was well mixed and left in the dark at 37°C. The absorbance value was measured at 517 nm after 30 min. Absolute ethanol instead of DPPH was taken as the control A₁, and absolute ethanol instead of sample solution was taken as the blank A₀.

The clearance rate of DPPH was calculated according to the following formula: DPPH clearance rate (%) = [1-(A-A₀)/A₁ ]×100%.

2.12. Determination of Amino Acids

Preparation of the standard solution: Each standard sample of a single amino acid was weighed in the same beaker, dissolved in 8.3 ml of 6 mol/L hydrochloric acid solution, and accurately transferred to a 250 mL volumetric flask. The standard reserve solution of amino acids was prepared by diluting the volume to the desired scale with water. A total of 1.0 mL of standard amino acid reserve solution was accurately transferred to a 10 ml volumetric flask, a pH 2.2 sodium citrate buffer solution was added, and then the solution was mixed; the resulting solution was the standard analysis liquid.

Samples were measured with ninhydrin, and the OD values of the samples were controlled between 0.1-1.5. Samples were filtered through a 0.45 µm membrane and transferred into liquid phase sample vials for measurement. An automatic amino acid analyzer (L-8900 basic amino acid analyzer: Hitachi High-tech Co., LTD., Tokyo, Japan) was used to detect amino acids based on the reference method [35].

2.13. Observation of the Lonicera japonica Internal Structure by Scanning Electron Microscopy

Sample preparation: The samples of L. japonica wine were absorbed as drops and placed on a quartz plate. The samples were dried completely at room temperature and sprayed with platinum to improve the conductivity of the samples.

2.14. Statistical Analysis

Each experiment was repeated three times. All the data are expressed as the mean ± SD (standard deviation). IBM SPSS Statistics was applied for statistical and variance (ANOVA) analysis with a significance level. Additionally, Graph Pad Prism 6.01 was used.

3. Results

3.1. GC-MS Analysis Results for Lonicera japonica Wine

GC-MS was used to identify the volatile flavor compounds (Table 1). These compounds included alcohols, esters, acids, aldehydes, ketones, and volatile compounds. The differences in flavor compounds between fermentation with residue and fermentation without residue were not obvious. The main metabolites consisted of eighteen types of esters, five alcohols, two aldehydes, six terpene classes, and four acids, while the composition of flavor substances after liquor extraction was lower, with eight esters, four alcohols, two aldehydes, two terpenes, and three acids. Table 1 analyzes the differences in the flavor substances of untreated L. japonica wine, including the following common substances: isoamyl caprylate, hexadecanoic acid ethyl ester, ethanol, 2-methylpropanol, 2-methylbutanol, benzene ethanol, acetal, benzaldehyde, transeugenene, imidodicarbonic acid, 2,4,5-trimethyl-1,3-dioxalane, acetic acid, 2,3-O-benzyl-d-mannosan, and thiosemicarbazone.

In terms of qualitative flavor compounds, L. japonica wine had the largest number of ester compounds, and these esters mostly came from the fermentation process that consisted of the esterification of alcohol with acid. Due to the high content of ethanol in liquor, more ethyl ester was formed through yeast fermentation, and this ethyl ester accounted for a large proportion of the ester.

The results of the physicochemical characterization of L. japonica wine after full brewing are shown in Table 2. As L. japonica itself contains a low sugar content, some exogenous food‐grade sugar was added in the brewing process, which raised the final alcohol content to approximately 17 vol% after fermentation.

There was no significant difference in the alcohol content of the experimental groups. The extracted alcohol level of L. japonica wine remained the same because liquor has no microorganisms; thus, the L. japonica broth did not undergo secondary fermentation, and the total acid content was higher with a low pH.

3.2. Polyphenol Comparison

The content of polyphenols in the samples was calculated through the use of ultraviolet spectrophotometry, which was based on the calibration curve of gallic acid and with the OD value of the sample measured at a wavelength of 765 nm. The change trend of the polyphenol content with increasing fermentation time was found to be linear (Figure 2, left Y).

Table 1. Main volatile flavor compounds identified in Lonicera japonica wine using GC-MS

Table 2. Detection of physiochemical indicators in Lonicera japonica wine

Figure 2. Relationship of polyphenols changes in Lonicera japonica wine at different fermentation time

The content of polyphenols reached the maximum value, and the content of polyphenols in L. japonica wine reached a satisfactory level. After the 30-day fermentation period, the polyphenol content increased significantly and exhibited the following values: fermentation without residue was 0.73±0.02 mg/mL, fermentation with residue was 0.61±0.03mg/mL, and liquor extraction was 0.45±0.03mg/mL. Figure 3 illustrates the statistical significance between the fermentation and extraction data. The content of polyphenols was clearly higher than that of unleavened broth, which might reflect the impact of the yeast strain on the phenolic extraction and bioconversion during fermentation by regulating their metabolism and interaction processes.

3.3. Comparison Results of Chlorogenic Acid

In the process of fermentation, the chlorogenic acid content was positively correlated with increasing fermentation time (Figure 3, left X). After fermentation was terminated, the concentration of chlorogenic acid in the fermented samples was significantly higher than the chlorogenic acid concentrations after liquor extraction and in the unfermented broth; the yield of chlorogenic acid increased by 1.85, 1.53, and 0.87 times (for fermentation with residue, fermentation without reside, and liquor extraction, respectively) compared with that of unfermented broth. Compared with direct extraction, yeast fermentation was more effective in improving the content of chlorogenic acid, and the rate of formation was also more rapid than that with liquor extraction. SPASS software was used to analyze the data of each group, and the results showed that the experimental data had statistical significance (Figure 4). Therefore, fermentation and extraction had significant effects on the composition and content of L. japonica wine.

Figure 3. Effects of different treatment methods on polyphenol content in Lonicera japonica wine. Different letters above the bars indicate significant difference according to least significant difference test at p<0.05

Figure 4. Effects of different treatment methods on chlorogenic acid content in Lonicera japonica wine. Different letters above the bars indicate significant difference according to least significant difference test at p<0.05

Figure 5. Comparison of ABTS and DPPH clearance rate among three wine samples in Lonicera japonica wine

3.4. Comparison of Antioxidant Activities

The linear correlation between the total polyphenols and antioxidant activity (ABTS and DPPH values) of the L. japonica wine was analyzed. The ability of polyphenols to scavenge free radicals of ABTS and DPPH in three types of L. japonica wines was observed (Figure 5). This indicated that the L. japonica broth could improve the rate that ABTS scavenges free radicals after yeast fermentation. The initial clearance rate of the raw broth was approximately 87%, and the three types of wines after fermentation reached 94%.

According to this characteristic, many cosmetic industries could use fermented L. japonica in certain amounts as a raw material in various products that may benefit from an increased antioxidant capacity to delay aging.

Our results suggested that the variations in the total polyphenol content and antioxidant activity among the L. japonica wines with yeast fermentation were distinct from those in the control group that underwent liquor extraction.

3.5. Results of the Amino Acid Analysis

The changes in amino acids in L. japonica wine during the brewing process were detected by an automatic amino acid analyzer (Table 3). Either the number of amino acid species sharply decreased or most of the amino acid content decreased, thereby showing that the amino acid type, content and production wine yeast microbes, were closely related. In the brewing process, in addition to Asp, Thr, Glu, Gly, Cys, Val, Tyr, and Lys, which are not used by yeast, the rest of the present amino acids would be consumed in a large amount by microbial activities, and the decarboxylation reduction reaction would occur; this reaction would result into corresponding advances in the wine and the production of some volatile esters. The amount of amino acids also clearly decreased in the liquor extraction control group because most of the free amino acids were soluble in ethanol, thus effectively solving the damage of ethanol to the human body. The decrease in free amino acids is beneficial for increasing the flavor of L. japonica wine. The next step was to further explore the specific metabolic process of amino acids under the action of yeast.

Table 3. Determination of free amino acid components (unit mg/L)

Figure 6. Morphological changes of Lonicera japonica wine by treatment with fermentation and extraction during the experiment, photos were taken at the same location. A represents Lonicera japonica solution without any treatment; B represents Lonicera japonica wine of fermentation without slag; C stands for Lonicera japonica wine of fermentation with slag; D represents Lonicera japonica wine of extraction by liquor. 1, 2 stands for observation using scanning electron microscope at 500, 50000x respectively

3.6. Lonicera japonica Degradation

L. japonica was degraded by yeast fermentation. The morphological structure of the four samples was observed by using scanning electron microscopy (Figure 6), which could directly show the difference of the samples before and after fermentation. Figure A shows the Lonicera japonica broth without fermentation. The L. japonica fibrous tissue was very tight, with no decomposition by external forces, and the whole surface structure was complete. Figures B and C show the L. japonica broth after fermentation. It could clearly be seen from the graph that the structure of the plant tissue had been destroyed and the internal organization of L. japonica was broken down to form a new structure. Therefore, we provided evidence of a new transformation produced by fermentation. Figure D shows that the plant tissue of L. japonica is not completely destroyed and becomes loose after liquor extraction.

These results demonstrate that the L. japonica broth would undergo changes in its internal structure after yeast fermentation because of the transformation of some enzymes. Thus, the more valuable substances in L. japonica were decomposed and incorporated into the wine.

4. Discussion

L. japonica was subjected to a fermentation treatment, which significantly increased the amount of active substances in the Lonicera japonica wine without affecting its original compounds. The production of L. japonica wine through yeast metabolism could produce many beneficial flavor substances (Figure 7).

On the other hand, the chlorogenic acid and total polyphenol contents in all the fermentation-treated L. japonica samples were significantly higher than those from the liquor extraction control group.

Polyphenols are very important molecules in many wines and are responsible for their quality and sensorial characteristics, such as taste and color. Compared with other wines with health benefits, the polyphenol concentration in Tower brand yellow wine was 0.544 mg/mL; Huizelong yellow rice wine was 0.303 mg/mL; and Kuaijishan was 0.430 mg/mL [36]. The total content of polyphenols in wine were closely related to the antioxidant capacity. Thus, the content of polyphenols affected the antioxidant capacity. Many studies have reported positive correlations between the total content of polyphenols and the antioxidant activity of different wines, including white, red, fruit and glutinous rice wines [37,38]. The results showed that the antioxidant activity of the Lonicera japonica wine reached a relatively high level.

Chlorogenic acid is the main active ingredient in L. japonica. Its leaching rate was very low in the initial test. After fermentation, this active ingredient of L. japonica was extracted, and its nutritional value was higher than that of simply drinking L. japonica tea. Throughout the fermentation process, the content of chlorogenic acid reached a maximum value and then started to decline until reaching a steady state. This result was satisfactory and demonstrated a certain theoretical basis for the medicinal value and extraction of chlorogenic acid. It could be further speculated that the increase in chlorogenic acid content was attributed to three major factors. One was that chlorogenic acid in plant cells was extracted by organic solvents such as ethanol produced during fermentation. The second was that chlorogenic acid was converted into shikimic acid by the addition of glucose under the catalysis of enzymes in plant cells. In this work, we identified a third factor in that a chlorogenic acid precursor compound was formed during fermentation. The new chlorogenic acid formed by the esterification, substitution, and condensation reactions of quinic acid and ferulic acid, caffeic acid, and shikimic acid was then converted to phenylalanine before a new chlorogenic acid was finally synthesized [39]. Furthermore, 3-O-feruloylquinic acid (3-FQA), 3,4-di-O-caffeoylquinic acid (3,4-CQA), and 3,5-di-O-caffeoylquinic acid (3,5-CQA) were metabolically produced [40,41]. These main secondary chlorogenic acid metabolites were one of the reasons for the increase in chlorogenic acid content. This result was verified by the literature in which 3,5-di-O-caffeoylquinic acid, 3-O-caffeoylquinic acid, 4,5-di-O-caffeoylquinic acid and 3,4-di-O-caffeoylquinic acid were reported to be the main secondary metabolites in suspension culture cells [42].

In addition, the L. japonica broth was treated at high temperature and pressure, and some active substances might be released during sterilization. It has been reported that when L. japonica is treated under high pressure, the contents of major phenolic acids and flavonoids, such as chlorogenic acid, caffeic acid and luteolin, significantly increase. This observation is probably due to the release of cell wall-bound phenolic compounds. The antioxidant activities of L. japonica were significantly increased after the high-pressure treatment [43]. Figure 8 shows the metabolic condensation reactions of quinic acid and caffeyl-CoA. It was first discovered that quinic acid, coenzyme A and ATP were essential for the chlorogenic acid synthesis pathway, and it was believed that chlorogenic acid would not be produced without quinic acid. Moreover, Stckigt et al. found that caffeyl-CoA was an inevitable intermediate in the chlorogenic acid synthesis pathway. Later, Ulbrich et al. isolated hydroxylated cinnamic acid-CoA:hydroxycinnamoyl-CoA (quinate hydroxycinnamoyl transferase, HQT) from two plant cells, and this enzyme could catalyze caffeyl-CoA and quinine to produce chlorogenic acid; thus, they believed that HQT was a key enzyme that directly produced chlorogenic acid.

The solubility of chlorogenic acid would increase in aqueous solution. Chlorogenic acid is soluble in ethanol, acetone, and methanol, slightly soluble in ethyl acetate, but hardly soluble in chloroform, ether, benzene and other lipophilic organic solvents. Chlorogenic acid is a polar organic acid that is not very stable, so it is easy to isomerize in the process of extraction or fermentation. Therefore, this study greatly improved the content of chlorogenic acid by fermenting L. japonica broth with yeast, and the medicinal value of L. japonica was correspondingly improved.

On the other hand, the content of amino acids in the L. japonica wine were mainly from the enzymatic hydrolysis of the raw material proteins during the fermentation of metabolites and the autolysis of yeast cells after fermentation [44,45,46]. During the fermentation of L. japonica, the transformation and formation of various compounds were related to the presence of certain amino acids. For the flavor analysis of the L. japonica wine, aromatic ester substances mainly presented a fruity aroma, which was most of the aromatic substance variety in flower wine. Most of the aromatic compounds were floral, which could enhance the aroma and coordination of the wine body. Additionally, these compounds mainly came from the catabolism of aromatic amino acids in the raw material. Due to the sugar and high carbon content in the raw materials used for producing the Lonicera japonica wine, the carbon atoms formed saturated hydrocarbon chains after yeast fermentation. There was little alcohol variety, which mainly stemmed from the conversion of sugars and amino acids in the fermentation stage

In addition, the L. japonica wine also contained some synthesized drug substances, namely, linalool, terpineol, and terpene oil compounds. This study showed that linalool was one of the major pharmacological substances in L. japonica, and as a functional active substance, linalool has been proven to reduce pulmonary inflammation [47]. Most terpenoids show inhibitory activity against HBsAg and HBeAg secretion and HBV DNA replication. These results indicated that L. japonica flower buds could serve as a functional food for antihepatoma and anti-HBV activities [48]. Thus, Lonicera japonica wine has potential for being applied in a health care role.

Figure 7. Main metabolites of the Lonicera japonica fermentation process and produces some flavor through yeast metabolism

Figure 8. Biochemestry formation pathway of chlorogenic acid

5. Conclusion

In this work, we developed a healthy functional wine through a new liquid fermentation method with a low alcohol content; furthermore, the flavor and active components in the L. japonica wine were analyzed.

The pharmacological composition and function of L. japonica showed that yeast fermentation increased the main nutritional components, namely, the contents of chlorogenic acid and polyphenols, the oxidation resistance activity, and the number of metabolites and flavor compounds. The L. japonica wine contained various esters, alcohols, aldehydes, terpene classes and acids. Another feature was that flowers were used as the raw material to avoid the production of methanol and fusel oil. Methanol and fusel oil are produced when grain is added as an auxiliary material, which is common in the production of conventional flower wine. Thus, the production of methanol was eliminated from the raw material, and the flavor compounds and active functional components were synthesized by yeast metabolism during fermentation. Therefore, the nutrients and active functional components in L. japonica and the flower flavor of L. japonica could be preserved. This suggested fermentation process is better than the liquor extraction process and is a safe and controllable metabolic process.

Author Contribution

Thanks to all authors for their contributions to this manuscript, Xinli Liu designed the research; Xiaolong Zhou, Wenting Ruan, Guoxiang Lin performed the experiments; Xiaolong Zhou analyzed the data and wrote this manuscript. In the end, Borras Orlando and Jing Zhao provided guidance and revision of the manuscript.

Disclosure Statement

The authors declare no conflicts of interest.

This study does not involve any human or animal testing.

Funding

This work was supported by the Shandong Province Key Research and Development Program (Medical Food and Major Scientific and Technological Innovation Project), 2018YYSP018, and the Yeast Beta Glucan Structure Nutrition Correlation and Carbohydrate Component Controllable Production Technology Research Project.

References

[1]   | Liu Q, Fang J, Wang P, Du Z, Li Y, Wang S, & Ding K. 2017. Characterization of a pectin from LonicerajaponicaThunb and its inhibition effect on Aβ42 aggregation and promotion of neuritogenesis. Int.J.Biol. Macromol: 107, 112.
[2]   | Wang D-Y, Zhao X-M, & Liu Y-L. 2017. Hypoglycemic and hypolipidemic effects of a polysaccharide from flower buds of Lonicera japonica in streptozotocin-induced diabetic rats.Int. J. Biol. Macromol. 102: 396-404.
[3]   | Jiang B, Hao Z-S, Wu W-D, Li Z-S, & Zhang J-R. 2018. A new PAT application: Optimization of processing methods for honeysuckle flower (LoniceraeJaponicaeFlos) and wild honeysuckle flower. Journal of Traditional Chinese Medical Sciences 7:199-205.
[4]   | Li J, Jin S-Y, Zu G-M, Luo W, Wang C-J, Zhao Y-J, & Fu Y-J. 2013. Rapid preparative extraction and determination of major organic acids in honeysuckle (Lonicera japonica Thunb.) tea.J. Food Anal 33:139-145.
[5]   | Liu Z-X, Cheng Z-Y, He Q-J, Lin B, Gao P-Y, Li L-Z, & Liu Q-B, Song S-J. 2016. Secondary metabolites from the flower buds of Lonicera japonica and their in vitro anti-diabetic activities. Fitoterapia 110:44-51.
[6]   | Han J, Lv Q-Y, Jin S-Y, Zhang T-T, Jin S-X, Li X-Y,&Yuan H-L. 2014. Comparison of anti-bacterial activity of three types of di-O-caffeoylquinic acids in Lonicera japonica flowers based on microcalorimetry. Chinese Journal of Natural Medicines 12(2): 108-113.
[7]   | Fan, Z-L , Li L , Bai X-L , Zhang H , Liu Q-R , Zhang H , Fu, Y-J, &Moyo R . 2019. Extraction optimization, antioxidant activity, and tyrosinase inhibitory capacity of polyphenols from Lonicera japonica. FOOD SCIENCE & NUTRITION 7(5): 1786-1794.
[8]   | Lee Y-S , Lee Y-J ,&Park, S-N .2018. Synergistic Antimicrobial Effect of Lonicera japonica and Magnolia obovata Extracts and Potential as a Plant-Derived Natural Preservative. JOURNAL OF MICROBIOLOGY AND BIOTECHNOLOGY 28(11): 1814-1822.
[9]   | Xin N, Li W , Li Y-J ,Ma X-K , & Fu Z-P . 2011. Study of antivirus, antibacteria and immune functions of Gaoreqing freeze-dried powder. JOURNAL OF MEDICINAL PLANTS RESEARCH 5(22): 5407-5412.
[10]   | PalikovaI ,Valentova K , Oborna I, &Ulrichova J . 2009. Protectivity of Blue Honeysuckle Extract against Oxidative Human Endothelial Cells and Rat Hepatocyte Damage. Journal of Agricultural and Food Chemistry 57(15): 6584-6589.

[11]  
Seo O-N, Kim G-S, Park S, Lee J-H, Kim Y-H, Lee W-S, Lee S-J, Kim C-Y, Jin J-S ,Choi S-K ,& Shin S-C. 2012. Determination of polyphenol components of Lonicera japonica Thunb. Using liquid chromatography–tandem mass spectrometry: contribution to the overall antioxidant activity. Food Chem 134 (1):572-577.

[12]   | Kwak W-J, Han C-K, Chang H-W, Kim H-P, Kang S-S, & Son K-H. 2003. Loniceroside C, an anti‐inflammatory saponins from Lonicera japonica. Chemical & Pharmaceutical Bulletin 51: 333-335.
[13]   | Shang X-F, Pan H, Li M-X, Miao X-L, &Ding H. 2011.Lonicera japonica Thunb.:Ethnopharmacology, phytochemistry and pharmacology of an important traditional Chinese medicine. Journal of Ethnopharmacology 138(1): 1-21.
[14]   | He L, & Chen S-L. 2013. Research progress of chlorogenic acid synthesis pathway in plants. Pharmaceutical biotechnology: 20(05): 463-466.
[15]   | RudkinG-O, & Nelson J-M. 1947. Chlorogenic acid and respiration of sweet potatoes. J Am Chem Soc 69: 1470.
[16]   | Shi L-S, Mu T-H, & Sun H-N. 2014. Advances in chlorogenic acids at home and abroad. Journal of nuclear agriculture 28(02): 292-301.
[17]   | Bassoli B-K, Cassolla P, & Borba-Murad G-R . 2008. Chlorogenic acid reduces the plasma glucose peak in the oral glucose tolerance test:effects on hepatic glucose release and glycaemia. Cell Biochemistry and Function 26 (3):320-328.
[18]   | Dos Santos M-D, Almeida M-C, & Lopes N-P . 2006. Evaluation of the anti-inflammatory, analgesic and antipyretic activities of the natural polyphenol chlorogenic acid.Biological and Pharmaceutical Bulletin 29 (11): 2236-2240.
[19]   | Yang X-F, Wang Y, & Ma Y-G. 2013. Extraction, separation and structural identification of chlorogenic acid from eucommiaulmoides leaves. Food research and development 34(04): 32-34.
[20]   | Watanabe T, Arai Y, Mitsui Y, Kusaura T, Okawa W, Kajihara Y , & Saito I. 2006. The blood pressure-lowering effect and safety of chlorogenic acid from green coffee bean extract in essential hypertension. Clinical and Experimental Hypertension 28(5): 439449.
[21]   | Wu Z-Y, Chen L-Y, Guo Z-H, Li K-Y, Fu Y-X, Zhu J-L, Chen X-T, Huang C, Zheng C-L, Ma Y-H, Li X-G, Zhou J, Wang Z-Z, Xiao W & Wang Y-H. Systems pharmacology uncovers serotonergic pathway mediated psychotherapeutic effects of Lonicerae Japonicae Flos. JOURNAL OF FUNCTIONAL FOODS 60.
[22]   | Rodriguez de Sotillo D-V, Hadley M, &Sotillo J-E. 2005. Insulin receptor exon 11+/− is expressed in Zucker (fa/fa) rats, and chlorogenic acid modifies their plasma insulin and liver protein and DNA.The Journal of Nutritional Biochemistry 17(1): 63-71.
[23]   | Nicasio P, Aguilar-Santamar A-L, &Arandae. 2005. Hypoglycemic effect and chlorogenic acid content in two Cecropia species. Phytotherapy Research 19 (8): 661-664.
[24]   | Muhammad N, Ghulam J-K, & Fang F-X. 2017. Chlorogenic acid (CGA): A pharmacological review and call for furtherresearch. Biomedicine & Pharmacotherapy 97:67-74.
[25]   | Lei W, Hai H-L, & Cao H-B. 2013. Development of multifunctional health wine of honeysuckle.Guangdong chemical industry 40(16): 29-30.
[26]   | Zou R, You Y-M, Chen Z-X, Hu K, & Ran L. (2016). Effects of drying method on polyphenols and antioxidant activity of honeysuckle. Food science 37(05):78-83.
[27]   | Ying G, Jun W-H, & Jian D. 2014. Study on brewing technology and stability of blue honeysuckle. Science and Technology of Food Industry: 2 206-209.
[28]   | Akkarachaneeyakorn S &Tinrat S. 2015. Effects of types and amounts of stabilizers on physical and sensory characteristics of cloudy ready-to-drink mulberry fruit juice. Food Science & Nutrition 3(3): 213-220.
[29]   | Wang C-Y, Liu Y-W, Jia J-Q, Sivakumar T-R, Fan T, &Gui Z-Z. 2013. Optimization of fermentation process for preparation of mulberry fruit wine by response surface methodology. African Journal of Microbiology Research 7: 227- 236.
[30]   | Wang L, Sun X, Li F, Yu D, Liu X, Huang W, & Zhan J. 2015. Dynamic changes in phenolic compounds, colour and antioxidant activity of mulberry wine during alcoholic fermentation. Journal of Functional Foods 18:254-265.
[31]   | Ivana G &Mirella N. 2015. Polyphenols content, phenolics profile and antioxidant activity of organic red wines produced without sulfur dioxide/sulfites addition in comparison to conventional red wines. Food Chemistry 179: 336-342.
[32]   | Wu Y-X, Liu B, Chang Y-L, & Wang Q. (2014). Optimization of Modified Supercritical CO2 Extraction of Chlorogenic Acid from the Flower Buds of Lonicera japonica Thunb and Determination of Antioxidant Activity of the Extracts. JOURNAL OF LIQUID CHROMATOGRAPHY & RELATED TECHNOLOGIES 38(4): 443-450.
[33]   | Re R, Pellegrini N, Proteggente A, Pannala A, Yang M, & Rice-Evans C. 1999. Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radical Biology and Medicine 26(9-10): 1231-1237.
[34]   | Yang B, Zhao M, Shi J, Yang N, & Jiang Y. 2008.Effect of ultrasonic treatment on the recovery and DPPH radical scavenging activity of polysaccharides from longan fruit pericarp. Food Chem 106(2): 685-690.
[35]   | LiJ-J, ZhaoA-P, LiD-M,& HeY. (2019). Comparative study of the free amino acid compositions and contents in three different botanical origins of Coptis herb. BIOCHEMICAL SYSTEMATICS AND ECOLOGY 83:117-120.
[36]   | Liu Y. 2016. Research on evaluation of health value of traditional Chinese yellow rice wine. University of jinan.
[37]   | Ljevar A, Curko N, Tomasevic M, Radosevic K Srcek V-G, Ganic K-K. 2016. Phenolic composition, antioxidant capacity and in vitro cytotoxicity assessment of fruit wines.Food Technology and Biotechnology 54(2): 145-155.
[38]   | Simonetti P, Pietta P, Testolin G. 1997. Polyphenol content and total antioxidant potential of selected Italian wines. Journal of Agricultural and Food Chemistry 45(4).
[39]   | Wang W-L, Wen C, Guo Q-P, Duan Y-H, Li Y-H, He S-P, & Li F-N. 2017. Bioactivity and mechanism of chlorogenic acid. Chinese journal of animal nutrition 29(07): 2220-2227.
[40]   | Meinhart A-D, Damin FM , Caldeirao L , Ferreira D-S, Tayse F, Teixeira J,& Godoy H-T .2017. Chlorogenic acid isomer contents in 100 plants commercialized in Brazil. Food Research International 99: 522-530.
[41]   | Muhammad N, Ghulam J-K, & Fang F-X. 2017. Chlorogenic acid (CGA): A pharmacological review and call for furtherresearch. Biomedicine & Pharmacotherapy 97:67-74.
[42]   | Hu M, Hu Z-J, Du L-D, Du J, Luo, Q-S, Xiong J-H. 2019. Establishment of cell suspension culture of Lonicera japonica Thunb and analysis its major secondary metabolites. INDUSTRIAL CROPS AND PRODUCTS137: 98-104.
[43]   | Lee J, Kang Y-R , Kim Y-J , & Chang Y-H. 2019. Effect of high pressure and treatment time on nutraceuticals and antioxidant properties of Lonicera japonica Thunb. INNOVATIVE FOOD SCIENCE & EMERGING TECHNOLOGIES 54:243-251.
[44]   | Soufleros E, Bouloumpasi E, Tsarchopoulos C, et al. 2003. Primary amino acid profiles of Greek white wines and their use in classification according to variety, origin and vintage. Food Chemistry 80(2): 261-273.
[45]   | Shen F, Niu X, Yang D, et al. 2010. Determination of amino acids in Chinese rice wine by fourier transform near-infrared spectroscopy. Journal of Agricultural and Food Chemistry 58 (17):9809-9816.
[46]   | Aquino F-W, Boso L-M, Cardoso D-R, et al. 2008.Amino acids profile of sugar cane spirit (cachaa), rum, and whisky. Food Chemistry 108 (2):784-793.
[47]   | Wu Q, Yu L, & Qiu J. 2014. Linalool attenuates lung inflammation induced by Pasteurellamultocida via activating Nrf-2 signaling pathway. International Immunopharmacology 21 (2): 456-463.
[48]   | Ge L-L, Xiao L-Y, Wan H-Q, Li J-M, Lv K-P, Peng S-S, Zhou B-P, Li T-Y, & Zeng X-B. 2019. Chemical constituents from Lonicera japonica flower buds and their anti-hepatoma and anti-HBV activities. BIOORGANIC CHEMISTRY.