Development of the aged (BLACK) garlic production process to obtain fermented garlic oil

Abstract

This invention is related to producing fermentation-aged (black) garlic from fresh Kastamonu Taskopru Garlic in a moisture-adjusted oven with a new method. The processing steps include; (A) Special polystyrene containers with ventilation lids, (B) Separation of freshly harvested inner and outer cloves of Taskopru Garlic, (C) Placing the garlic cloves in the special containers that allow ventilation for fermentation, (D) The aged garlic was obtained due to the fermentation of fresh garlic.

Description

[0001] DEVELOPMENT OF THE AGED (BLACK) GARLIC PRODUCTION PROCESS TO OBTAIN FERMENTED GARLIC OIL

[0002] TECHNICAL FIELD

[0003] This invention is related to producing fermentation-aged (black) garlic from fresh Kastamonu Taskopru Garlic in a moisture-adjusted oven with a new method. The processing steps include; (A) Special polystyrene containers with ventilation lids, (B) Separation of freshly harvested inner and outer cloves of Taskopru Garlic, (C) Placing the garlic cloves in the special containers that allow ventilation for fermentation, (D) The aged garlic was obtained due to the fermentation of fresh garlic.

[0004] THE PREVIOUS TECHNIQUE

[0005] Taskopru Garlic, one of the geographical indication products of the European Union, is one of the local agricultural products grown in natural agriculture with high-quality natural garlic in Taskopru district of Kastamonu province and widely grown.

[0006] There are many companies operating in this field in Kastamonu / Turkiye, and some companies produce also aged garlic. However, there are no products on the market for production, marketing, research&development activities related to the essential oil components of aged garlic.

[0007] No oil could be obtained from commercially sold aged garlic that has taken its place on the shelves. At the same time, essential oils were obtained at a meager yield of 0.0017% (50 pl / 3 kg of ripe garlic) from products that had just been completed fermentation and had not yet been packaged. Similar results have been obtained from the information received from the commercial production organizations and also the productions performed in the high-humidity cabinet in the laboratory environment. Then, the aged garlic and its essential oil components, obtained in the laboratory, were analyzed. As a result of these analyses, it was determined that almost all of the volatile components of fresh (white) garlic were lost, and the amount of beneficial sulfurous components remained at a trace level. However, it was determined that harmful products known to have toxic effects were also formed in significant quantities. This situation was interpreted as the chemical transformations progressing in a way that would give undesirable products due to the differences in environmental conditions, especially high temperature and incubation period, applied during the production of aged garlic. In both the aged garlic samples that we produce ourselves using the method used in the market and those obtained from the market, it was determined that the quality changes depending on the shelf life. It was also found that fermentation continues regardless of storage conditions (cold environment, etc.), and the amount of harmful components in the aged garlic content increases. In the current methods of aged garlic production, the garlic heads are subjected to fermentation without being separated into cloves. The fermentation process is monitored by tasting the cloves lined up outside the garlic heads. In this case, the inner teeth mature earlier and transform at a higher rate at the end of the process. As a result, it also contains more harmful ingredients, resulting in bitterness and an unpleasant aroma in the taste.

[0008] It was determined that these problems should have been mentioned in the interviews with the manufacturers of some aged garlic products on the market, and they only have problems with packaging. The web pages of companies working in this field today have been observed to contain misleading information about aged garlic and its content. Although it has been shown in vitro cell cultures (conducted by us) that aged garlic does not provide a therapeutic effect in cancer cell lines, companies that it is suitable for fighting cancer.

[0009] To solve the identified problems, several production methods were tested for the fermentation process of garlic, and the aged garlic production process was carried out, which contained the highest functional components and obtained essential oil with the highest efficiency.

[0010] Garlic can be used in different forms and different areas, such as fresh or aged garlic, smoked garlic, garlic oil, macerate, extract, powder, supplement pill, garlic juice, alcoholic tincture, etc. Aged garlic is preferred because it does not have an intense, unpleasant flavor due to its reduced allicin content compared to fresh garlic. Aged garlic, which has been proven to have high antioxidant capacity, is a product that has been focused on a lot in recent years.

[0011] In the aged garlic production process, fresh-white garlic is fermented for a certain period under controlled heat and humidity conditions. In this process, polyphenolic compounds appear as a result of the Maillard reaction occurring in garlic, and a completely additive-free new product is obtained from fresh garlic. It is known that the Maillard reaction improves the antioxidant-based bioactive properties of aged garlic. Changing the temperature (40-90 °C), humidity (50-100%), and waiting time (10-80 days) used for aged garlic production also changes the physicochemical properties of the product obtained. When fermentation is applied at high temperatures, the aging period of garlic is shortened. When the aging process is carried out at 70 °C, the aging speed is twice as fast as at 60 °C. In aged garlic produced faster at higher temperatures (90 °C), non-ideal flavors such as bitter and sour occur. However, since the color of garlic does not turn completely black during the aging process, which is carried out at a low temperature (60°C), this temperature is not considered suitable for aging. The quality of aged garlic is affected by temperature and other factors such as humidity and time. Therefore, further research is necessary. It has been stated that oils obtained from garlic grown in different geographical regions show qualitative similarities. Still, there are quantitative differences in the concentrations of organosulfur compounds, which probably affect garlic's medicinal and organoleptic properties.

[0012] General Production Process of Commercially Aged Garlic

[0013] In standard aged garlic production methods, fresh garlic is placed in a humidity- controlled fermentation device, usually wrapped in aluminum foil, without peeling. The aluminum foil is tightly covered to prevent contamination and odor. It is kept in the device for 20-40 days at a controlled constant temperature (70-90°C) and humidity (50-90%) until the desired level of blackening, taste, and aroma characteristics are obtained. Fermented (aged) garlic is generally produced in the market by this method, and there are differences in temperature and humidity values by the company. However, temperatures above 65°C are usually preferred to be faster in commercial production, and 70°C is most used.

[0014] DESCRIPTION OF THE INVENTION

[0015] Garlic (Allium sativum L.) has been accepted as an effective and natural therapeutic agent in the field of health for many years due to its various bioactive properties such as antimicrobial (antibacterial-antifungal-antiviral), hepatoprotective, antioxidant, anti-inflammatory, fibrinolytic, anti-platelet aggregation activity. However, studies on aged garlic, especially its therapeutic activity, are still insufficient. The new method developed in this invention has the following advantages.

[0016] - With the developed aged garlic production process, garlic's inner and outer cloves are fermented separately in special containers. In this way, the undesirable advanced fermentation of the garlic cloves arranged in the inner row is prevented.

[0017] - The garlic cloves arranged in the inner row, richer in volatile components, mature earlier than the outer garlic cloves and become ready for use.

[0018] In the fermentation process of garlic, special containers made of polystyrene, with ventilation lids and no toxic effect on mammalian cells, are used instead of aluminum foil. In this way, volatile components remain in the environment for longer, and fermentation progresses faster without the formation of harmful components.

[0019] - Since aluminum-containing foils that are toxic to mammalian creatures are not used, aluminum and related toxicity can not be leak into garlic.

[0020] - The volatile garlic oil produced contains components with known antioxidant properties and sulfurous components that support cellular regeneration at a higher rate than their counterparts.

[0021] As a result, the ability to produce volatile garlic oil as a raw material from ripened garlic produced by fermentation from Ta§kdpru garlic (Kastamonu-Turkey), one of the geographically registered products of the European Union, will allow the determination of different usage potentials of this product in different sectors (pharmaceutical industry, biomaterial, nutritional supplement, etc.).

[0022] Figures List

[0023] Figure 1. Chromatogram and mass spectrometry graphs (GC-MS spectra) show that there is a single volatile compound in the aqueous distillate of the outer cloves (1 a) and inner cloves (1 b) of commercial aged garlic.

[0024] Figure 2. Chromatogram obtained from analyzing volatile substances obtained directly from aged garlic by SPME technique using GC-MS.

[0025] Figure 3. Chromatogram obtained from the GC-MS analysis of the volatile oil from internal cloves of aged garlic.

[0026] Figure 4. Chromatogram obtained from the GC-MS analysis of the volatile oil from external cloves of aged garlic. Figure 5. Graph showing the distribution of MAS and HAS scores of the subjects according to the time points.

[0027] Figure 6. The graphs show changes in fibrinolytic activity parameters in serum samples on days 3-14-28 compared to day 0 of the control group subjects (KS) in which serum physiologic was applied.

[0028] (6a) tPA graph of control serum

[0029] (6b) PAI-1 graph of control serum

[0030] Figure 7a. Changes in fibrinolytic activity parameters in serum samples on days 3- 14-28 in the aged garlic essential oil (SSU) group compared to day 0 serum samples

[0031] (7a) tPA graph of SSU group

[0032] (7b) PAI-1 graph of SSU group

[0033] DETAILED DESCRIPTION OF THE INVENTION

[0034] In this detailed description, the alternatives of the aged garlic production process to obtain fermented garlic oil containing functional components for health with high efficiency, which can be used in research and development studies of health and pharmaceutical sciences, the subject of the invention, are explained.

[0035] The production process of aged garlic by fermenting fresh Taskopru Garlic in a humidity-controlled oven with a new method (A) Special polystyrene containers with ventilation lids, (B) Separating freshly harvested Ta§kdpru garlic and inner and outer cloves, (C) Placing the inner or outer garlic cloves in that special containers separately (D) Aged garlic obtained as a result of fermentation.

[0036] Essential Oil Extraction:

[0037] The aged garlic essential oil components are extracted with the standard Clevenger assembly used for oils heavier than water.

[0038] 1. Production Process for Obtaining Oil from Aged Garlic

[0039] The high-quality fresh garlic grown with natural agriculture in Taskopru-Kastamonu (in Turkiye) and having geographical indication by the Turkish Patent Institute and the European Union was fermented at different temperatures in a high-temperature humidity cabinet, and aged garlic production was carried out. The production time and components were determined. The extraction of the essential oil and fixed oil components of the aged garlic produced by the new method was carried out by conventional chemical methods and the characterization of the products was carried out.

[0040] The following steps were carried out within the scope of the new process: a. Ta§kdpru Garlic, purchased as medium clove size from a producer with a geographical registration certificate, was used. b. Garlic was used by separating it into cloves instead of being fermented as a whole head. At this stage, the clustered cloves inside the garlic head and the larger outer clustered cloves were separated from each other. c. In the fermentation process of garlic, polystyrene-made T-cell culture containers with ventilation lids, used for in vitro cell culture, were preferred. d. The shelled garlic cloves were placed in T containers, the inner and outer teeth separately, and the lids were closed. S e. The process was started by labeling the T containers loaded with garlic and placing them in a high-temperature humidity cabinet. f. The first 7 to 10 days of the process continued at 65-70 ±2 °C and the following days at 60-65 ±2 °C with a humidity of 90 ±5%. The inner cloves of garlic fermented to the desired level in 22-28 days, and the outer cloves fermented in 25-32 days. g. The flavor of the produced aged garlic was organoleptically tested. h. One day after production, the essential oil components of the aged garlic (the most crucial bioactive chemical components that give garlic its characteristics) were extracted and purified by water vapor distillation in the Clevenger assembly. i. Yield calculations were made. j. Samples of the product were analyzed by Gas Chromatography-Mass Spectroscopy (GC-MS).

[0041] 1.1. Detailed Production Process

[0042] Medium clove-sized Taskopru Garlic was used for the production of aged garlic. The fresh garlic was cleaned from its stems without separating its peel. The cloves arranged on the outside of the garlic bulb were separated, and the cloves arranged in clusters on the inside of the garlic bulb were separated from the bulb and grouped. Polystyrene containers with ventilation lids were used to minimize the loss of volatile substances in producing aged garlic and to shorten the fermentation time by retaining moisture more. The containers were filled entirely to the highest possible filling level at this stage. After being placed in the high-temperature humidity cabinet, it was left for fermentation at 65-70 ±2 °C for the first 7 to 10 days and at 60-65 ±2 °C for the following days in a >90% humidity environment. After being placed in a high-temperature humidity cabinet, it was left for fermentation at 65-70±2 °C for the first 7 to 10 days and at 60-65±2 °C for the following days in a >90% humidity environment. At this stage, maximum attention was paid to maintaining humidity. It has been observed that the fermentation time of fresh garlic varies with the waiting time (dryness rate) after harvest. At the end of the period, the aged garlic obtained by completing fermentation was stored at 4 °C for use in later processes. Cold storage was preferred because it slowed down the selfadvanced fermentation process of aged garlic.

[0043] 1.2. Extraction of Aged Garlic Essential Essential Oil Components

[0044] Garlic essential oil can be obtained by steam distillation methods. In the conducted studies, steam distillation methods were used with the help of the Clevenger assembly used for oils heavier than water to obtain oil from aged garlic (Technical Drawing And Explanatory Diagrams, Essential Oil Extraction). The commercial production process was changed so the volatile oil components could be isolated from the aged garlic. At the end of the fermentation process, if necessary, the aged garlic material was kept at +4°C for maximum one day, then taken directly to the Clevenger system to extract the volatile oil components. Then the extracted volatile aged garlic oil was analyzed by chromatographic methods. For this purpose,

[0045] - The aged garlic was taken into a Clevenger assembly (5L capacity ) with an amount of 3 kg with peels and cut into small pieces with the help of a mechanical grinder for 5±2 minutes.

[0046] - The ground garlic was transferred to the rodage balloon (5 L volume).

[0047] - After adding about 1.5 liters of deionized water to it, the balloon was placed on the heater.

[0048] - Then, the Clevenger assembly was installed, and the back cooler was connected to the water flow.

[0049] - Hydro-distillation was performed until no essential oil came from the oil collection column.

[0050] - The yield of essential oil was calculated according to Equation 1 . - Thanks to this change in the aged garlic production method, an average of 450 pl of matured garlic essential oil could be obtained from 3 kg of matured garlic with a yield of about 0.015%.

[0051] - Characterization was made by laboratory analyses.

[0052] - The chemical composition of the extracted aged garlic essential oil was determined by analyzing it with GC-MS.

[0053] Equation 1

[0054] 2. ANALYSIS

[0055] 2.1. Essential Oil Composition

[0056] Analysis of the components of essential oils diluted in methanol or hexane at the appropriate rate were analyzed with the GC-MS device with HP 5MS capillary column (Shimadzu® GCMS-QP2010 SE). The identification of the peaks obtained as a result of qualitative analysis for identifying the components was carried out by scanning the NIST and Wiley libraries of the GC-MS system. The essential oil components could not be obtained from the aged garlic produced by the commercial method; only aromatic water was obtained. A sample of aromatic water produced by water vapor distillation of aged garlic with the idea that there may be volatile component residues in it was also analyzed by GC-Ms. The following parameters were used in the GC-MS analyses:

[0057] - Column: RTX-5MS Capillary column (30 m; 0.25mm; 0.25 / zm).

[0058] - Carrier gas: Helium

[0059] - Column oven temperature: 40C

[0060] - Injection temperature: 250C

[0061] - Pressure: 100 kPa

[0062] - Injection mode: split

[0063] - Split ratio: 10

[0064] - Injection volume: 1 ul

[0065] - Oven temperature program: 3 min at 40 °C, 40 °C to 240 °C with a 4 °C / min increase, 10 min at 240 °C, a total of 63 min.

[0066] - Interface temperature: 250 °C

[0067] - Ion source temperature: 200 °C

[0068] - Total flow: 0.3000 mL / min

[0069] - B.Conc: 50.0% - Nebulization gas flow: 3.0 L / min

[0070] - Drying gas flow: 15.0 L / min

[0071] - DL temperature: 250C

[0072] - Heat block temperature: 400 °C

[0073] - CID Gas: 230 kPa

[0074] 2.2. Analysis of Total Volatile Compounds of Commercially Produced Aged Garlic Sample

[0075] Since essential oil could not be obtained from aged garlic, to understand whether there was any essential oil compound left in the aged garlic due to fermentation conditions at the end of the fermentation period, the volatile gas released from the aged garlic was collected in a fiber (Restek Pal SPME Fibers®). For this purpose, 80°C temperature was applied directly for 20 minutes in a closed bottle using the Solid-Phase Microextraction Method (SPME). After the content of this fiber was manually injected into the GC-MS device, analysis was performed with a GC-MS device with an HP-5MS capillary column.

[0076] 2.3. In Vivo Experiments on the Use of Essential Components of Aged Garlic as a Pharmaceutical Agent in the Surgical Field

[0077] 2.3.1. Experimental Animals

[0078] A total of 2 study groups were randomly generated from the subjects, each of which had 6 subjects. In the control group, one group (n=6) applied physiological serum (SF), and another group (n=6) applied only aged garlic essential oil (SSU).

[0079] 2.3.1.1. Creation of abrasion on cecum and application of film samples

[0080] - On day 0, general anesthesia was applied to the rats before the surgical operation for in vivo experiments. For this purpose, an anesthetic combination (ketamine HCI 50 mg / kg + xylazine HCI 10 mg / kg at a dose of 250 mg / kg) was injected intraperitoneally into the animals (Diehl et al., 2001 ).

[0081] - The hair on the abdominal area of the animals was shaved with an electric shaver and cleaned.

[0082] - Then, blood samples were taken from vena jugularis (Diehl et al. , 2001 ) to Ethylenediamine Tetraacetic Acid (EDTA) and gel blood tubes for hemogram and biochemical analyses.

[0083] - The subject was fixed in the dorsal position on the operating table with the help of a piece of medical tape. Iodized antiseptic, and then alcohol was applied to the shaved area. - The subject was covered with a sterile drape except for the abdominal area.

[0084] - A 2 cm long incision was made in the umbilical region with the help of a scalpel.

[0085] - After abdominal exploration, the cecum was pulled out and placed on the sterile drape.

[0086] - Abrasion was applied with sterile gauze until bleeding was observed to form an experimental adhesion lesion on the antimesenteric surface of the posterior cecum.

[0087] - Intraabdominal 0.5 ml isotonic sodium chloride (NaCI- 0.9%) was administered to all subjects (Diehl et al., 2001 ).

[0088] - Then, according to the study group (SF / SSU), the relevant material was applied topically on the adhesion area, and the cecum was placed in its normal intraabdominal position. Applications made to the cecum were photographed in all subjects.

[0089] - Muscles and peritoneal membrane were sutured with interrupted cruciate (X) sutures using absorbable suture material (Surgicryl® polyglactin 3 / 0, SMI Steinerberg-Belgium).

[0090] - The skin incision was closed with horizontal U sutures using silk thread (Ipek 3 / 0, Dogsan Medical Materials Industry Inc., Trabzon-Turkiye).

[0091] - After the incision line was closed by suturing, an iodized antiseptic was applied to the suture line.

[0092] - Each subject who underwent a surgical operation was taken to a separate clean cage and laid in a lateral position with their heads and necks straight. They were expected to wake up from anesthesia.

[0093] - During the postoperative period, the general condition of the animals, their mobility, feed, water consumption, respiration, urination, defecation, feces, and the condition of the operation wounds were observed and monitored at regular intervals, four times a day for the first three days and two times a day for the following days.

[0094] 2.3.1.2. Macroscopic adhesion scoring (MAS)

[0095] To examine the adhesion inhibitory efficacy of physiological serum (control) and aged garlic essential oil (SSU), which were topically applied to the abrasion surfaces of the cecum on experimental abrasion lesions on day 0 of the study, macroscopic evaluations were made according to Table 1 on postoperative days 3, 14, 28 and Macroscopic Adhesion Scores (MAS) were determined. In cases where adhesion was formed, tissue samples were taken and stored for histopathological and immunohistochemical evaluations and HAS scoring.

[0096] Macroscopic assessments made on postoperative days 3-14-28;

[0097] - Two subjects from each study group were placed under general anesthesia with the same preoperative procedures performed each study day.

[0098] - Subsequently, blood samples were taken, abdominal exploration was performed after re-laparotomy, the anti-mesenteric surfaces of the abraded ceca were examined, and postoperative adhesion was evaluated.

[0099] - The ceca of all subjects were photographed and recorded.

[0100] - At this stage, in cases where adhesions were formed, scoring was performed using the Blauer and Collins (1988) criteria (Table 1 ), and Macroscopic Adhesion Scores (MAS) were determined and recorded.

[0101] - In addition, tissue samples were taken from cases in which adhesion tissue formed and fixed in 10% formalin solution for histopathological and immunohistochemical evaluations.

[0102] Table 1. Macroscopic and histopathological intra-abdominal adhesion scoring criteria

[0103] 2.3.1.3. Hemogram and biochemical blood analyses

[0104] Blood samples were taken from the jugular vein of the experimental animals on preoperative (0) and postoperative days (3-14-28) under general anesthesia. They were examined by a fully automatic hemogram (Mindray® 2800 BC) and biochemical analyzer (Fuji® Dri-Chem NX500). In biochemical analyses, Total Protein (TP), Alkaline Phosphatase (ALP), Glucose (GLU), Glutamic Pyruvic Transaminase (GPT)ZAIanine Aminotransferase (ALT), Creatinine (CRE), Blood Urea Nitrogen (BUN) values were measured in the blood serum. The results of the blood analyses taken from the subjects at all time points were transferred to the computer as an Excel file for statistical evaluations and archived.

[0105] 2.3.1.4. Histopathological Adhesion Scoring (HAS)

[0106] Tissue samples taken from cases with macroscopic adhesion formation were scored using the criteria of Yilmaz et al. (2005) (Table 1 ) in histopathological examinations. Histopathological Adhesion Scores (HAS) were determined and recorded.

[0107] 2.3.1.5. Serological Evaluation of Fibrinolytic Activity

[0108] Blood serum obtained from blood samples taken from experimental animals on preoperative (0) and postoperative days (3-14-28) was also used to evaluate fibrinogen activity. Serum samples taken for this purpose were kept at -80°C before examination. Before the procedure, the temperature of all reagents and materials was waited until the room temperature of +25°C. After the serum samples were thawed, they were centrifuged at 2000 rpm for 20 minutes. In the evaluations, plasminogen activator inhibitor type 1 (PAI-1 ) and tissue plasminogen activator (tPA) levels were determined by ELISA method [Sunred® Rat Tissue PAI-1 ELISA Catalog Number: SRB-T-81738 (Lot Number: 202108) and Sunred® Rat t-PA ELISA Catalog Number: SRB-T-81108 (Lot Number: 202102)] following the kit protocols. Depending on the color intensity obtained at the end of the reaction, the absorbance values were measured at 450 nm using a microplate reader (Synergy® HTX MultiMode Reader, Biotek Instruments USA). Data regarding fibrinolysis parameters were evaluated by transferring them to graphs.

[0109] 3. RESULTS

[0110] 3.1. Essential Compound Findings in Aged Garlic Produced by Commercial Process

[0111] Essential oil could not be obtained from aged garlic produced by commercial methods. Still, volatile substance analysis was also performed on the aqueous distillate obtained to see if a volatile substance existed. It was found that only a single compound and, therefore, a single peak was found in the chromatograms (Figure 1 ). This compound was 2-furan carboxy aldehyde, also known as furfural. This volatile liquid compound is soluble in water, denser than water, colorless or reddish brown when exposed to light and air, and has a penetrating almond-like odor. It is a product of the Maillard reaction and acts as a metabolite. Chemically, it is an aldehyde that is a member of Furans. It can be toxic if ingested, absorbed through the skin, or inhaled (pubchem.ncbi.nlm.nih.gov, 2021 ). Since the aged garlic fermentation process is a long-term process under heat and humidity, it is a natural situation that causes the formation of this reaction product since the conditions required for the Maillard reaction to occur are provided.

[0112] After the inability to obtain essential oil from the aged garlic, SPME analysis was performed with the GC-MS device to determine whether there was an essential oil compound left in black garlic due to the fermentation conditions at the end of the fermentation period. The related chromatogram is given in Figure 2, and the analysis results are in Table 2.

[0113] It is seen in Table 2 that there are 54 different compounds in the gas released by heating ripe garlic, and most of them are not essential oil compounds but volatile long-chain hydrocarbons (alkane series). In addition, only 4 of the 17 essential oil compounds detected in the essential oil of white garlic were detected in the gas obtained directly by heating the aged garlic. However, diallyl trisulfide, which is the source of most of the pharmacological effects of the essential oil of white garlic, could not be determined in this gas, and the amount of diallyl disulfide, another important active ingredient of the essential oil of fresh garlic, was detected in the gas obtained directly from aged garlic at a rate of only 1 .55%.

[0114] These decreases in the specified parameters explain the inability to obtain essential oil under the analysis conditions by the Clevenger method. Similarly, it has been mentioned in recent studies that many volatile components are removed with prolonged high-temperature application by fermentation, and some volatile sulfur components detected in fresh garlic cannot be detected in aged garlic.

[0115] In the study of Molina-Calle et al. (2017), the ratios of diallyl disulfide, diallyl trisulfide and diallyl tetrasulfide among the essential components of fresh garlic were found to be 21 %, 21.3% and 0.21 %, respectively (the amounts of these compounds are much higher in Taskopru Garlic); while these values were found to be 0.84%, 0.86% and 0.11 % in aged garlic, respectively. Table 2. Results of analysis of volatiles obtained directly from commercially produced aged garlic by SPME technique. 3.2. Volatile Compound Analysis of Aged Garlic Obtained by New Production Process

[0116] The chromatograms obtained from qualitative analysis of the components of volatile oils obtained from aged garlic are given in Figure 3 (inner cloves) and Figure 4 (outer cloves). The volatile compound composition of the essential compound oil obtained from aged garlic from inner cloves is given in Table 3, and the volatile compound composition of the essential compound oil obtained from outer cloves is presented in Table 4. In the aged garlic essential oil of the garlic cloves in the inner row, 21 compounds that are not in fresh garlic essential oil, and in the aged garlic essential oil of the garlic cloves in the outer row, 18 compounds that are not in fresh garlic essential oil were detected. In the aged garlic essential oil obtained from the inner and outer rows of cloves, 12 compounds found in the essential oil of fresh garlic were detected (those written in dark black in Tables 3 and 4). The ratio of total diallyl sulfites with positive health effects in fresh garlic oil was 87.32%, and the ratio of total trisulfites was 54.83%. However, the ratio of total diallyl sulfites and total trisulfites in the aged garlic essential oil obtained from the inner cloves were determined as 30.69% and 42.51 %, respectively. The ratio of total diallyl sulfites and total tri-sulfites in the aged garlic essential oil obtained from the outer cloves was determined as 30.65% and 42.55%, respectively

[0117] According to the results, the aged garlic formation process also caused a significant decrease in the amounts of total diallyl sulfides and total trisulfides. In addition, the ratios of total diallyl sulfide and total trisulfide belonging to the aged garlic essential oils belonging to the inner and outer cloves are not significantly different from each other. The compounds found in aged garlic essential oils but not in fresh garlic essential oil, formed due to the aged garlic production process, are generally sulfur aroma compounds and are naturally found in the structure of some plants. Table 3. Volatile compound composition of aged garlic from inner cloves

[0118] Compounds numbered 6, 7, 9, 12, 13, 15, 16, 19, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, and 33 are compounds found in the essential oil of internally aged garlic but not in the essential oil of fresh garlic. Table 4. Volatile compound composition of aged garlic essential oil from outer cloves essential oil of aged garlic from internal cloves. Compounds numbered 12, 13, 19, 20, 21, 24, 25, 28, and 30 are compounds found only in the essential oil of aged garlic from outer cloves and are not detected in the essential oils of fresh garlic and aged garlic of internal cloves.

[0119] 3.3. In Vivo Analysis Results of Aged Garlic Essential Oil as an Intraabdominal Adhesion Prevention Agent 3.3.1. Clinical Assesment

[0120] No complications were observed regarding the general condition, condition of operation wounds, mobility, respiration, feed and water consumption, urination, and defecation of experimental animals used within the scope of in vivo experiments after the first 6 hours in the postoperative process. No postoperative complications were observed, such as opening, bleeding, etc., that would require intervention in the wounds of animals undergoing surgery.

[0121] 3.3.2. MAS and HAS

[0122] It was stated that the evaluation of the success criterion in terms of macroscopic adhesion formation was evaluated as successful if the macroscopic adhesion scores in the study groups were below 20% [that is, the subjects who scored 1 and below according to the criteria of Blauer and Collins (1988)].

[0123] In this context, the success criterion percentage values made according to the MAS results in the project subjects are given in Table 5.

[0124] Table 5. Success criterion values in the study groups according to the MAS results (%)

[0125] The adhesion tissue samples were evaluated with histochemical staining hematoxylin-eosin, safranin-O, and alcian blue staining. The staining results were assessed according to the criteria of Yilmaz et al. (2005) (Yilmaz et al., 2005) (Table 1 ). MAS and HAS levels were determined by scoring (Table 6). In histopathological evaluation, especially in samples containing aged garlic essential oil, adhesion formation was observed as HAS2 and HAS3. The results are presented in Table 6 and Figure 5.

[0126] Table 6. The distribution of macroscopic adhesion scoring (MAS) and histopathological adhesion scoring (HAS) subjects according to study groups and time points

[0127] 3.3.3. Blood Analysis

[0128] The average values of some parameters related to hemogram and biochemical analyses performed on blood samples taken from experimental animals at all time points are presented in Table 7. The obtained data showed that white blood cells were very high on day 0 in some subjects, but in blood samples taken at postoperative time points, white blood cells were determined to be at similar levels within normal limits. While red blood cell counts were also found to be low on day 0 in some subjects, it was found that there was an increase in red blood cell counts at postoperative time points and that they multiplied at similar rates in all subjects. It was observed that platelets, which play a role in coagulation, generally increased on postoperative day 3. However, at later time points, they decreased in aged garlic oil. It was found that blood alkaline phosphatase activity increased on postoperative day 3 but increased to preoperative levels at later time points.

[0129] Table 7. Mean values and standard error values of some blood parameters according to time points (preoperative day 0, postoperative days 3-14-28) 3.3.1. Serological Assessment of Fibrinolytic Activity

[0130] In vivo experiments, when the ELISA results of the samples of the control group subjects (KS) to whom physiological serum was applied to the cecal abrasion surface were compared with the ELISA results on days 3-14-28; it was seen that tPA expression was almost at the same level in CS3 and decreased in all other samples. When PAI-1 expressions were compared, it was seen that there was a decrease in PAI-1 value in the CS5 sample, and an increase was detected in the other samples (Figure 6). In addition, they were found to be compatible with MAS values. In light of these data, CS1 , CS2, CS4, and CS6 samples were found to be risky in terms of adhesion development.

[0131] In vivo experiments, when the ELISA results of the samples of subjects (SSU) to whom aged garlic essential oil was applied to the cecal abrasion surface were compared with the ELISA results of days 3-14-28, it was determined that tPA expression was decreased in SSU1 , SSU2, and SSU3 samples, and increased in SSU4, SSU5 and SSU6 samples. When PAI-1 expressions were compared, it was found to be increased in SSU3 and SSU4 samples and decreased in SSU1 , SSU2, SSU5, and SSU6 samples (Figure 7).

Claims

CLAIMS1 . The invention is a method of developing the matured (black) garlic production process to obtain fermented garlic oil, the characteristic of which is; i. Separating the inner and outer garlic cloves and placing them in T- culture containers with ventilation lids and closing the lids, ii. Placing the garlic-loaded T containers in a high-temperature and humidity cabinet, iii. Fermentation continues at 65-70 ±2 °C for the first 7-10 days and at 60-65 ±2 °C for the following days at 90 ±5% humidity, iv. The fermentation process of garlic cloves lined up in the inner row of the garlic bulb takes about 22-28 days, and garlic cloves lined up in the outer row takes about 25-32 days, v. The aged garlic is taken in an amount of 3 kg with peels and cut into small pieces with the help of a mechanical grinder for 5±2 minutes, vi. Transferring the ground garlic to a balloon with a rod of 5 L volume and placing the balloon on the heater after adding about 1 .5 L of deionized water, vii. The Clevenger assembly is installed on it, and the back cooler is connected to the water flow, viii. Performing hydrodistillation until no essential oil comes from the fat collection column, ix. One day after the fermentation process is completed, the volatile oil components of the aged garlic are purified by steam distillation extraction in the Clevenger assembly.

2. The invention is according to Claim 1 , and its feature is the use of polystyrene containers with ventilation lids, which minimize the loss of volatile substances in the production of aged garlic and reduce the fermentation time by retaining moisture more. Thereby reducing the number of harmful components while protecting useful chemical components more.

3. The invention according to Claim 1 , and the feature is that the cold storage method is used as it enables the aged garlic to slow down the further fermentation process on its own.

4. The invention is according to Claim 1 , and its feature is that at the end of the fermentation process, the aged garlic material is kept at +4°C for a maximum of one day if deemed necessary for the completion of the preparations and then taken directly to the Clevenger system to extract the essential oil components.

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