A Mini Review on Complete Study of Black Garlic: Extraction Techniques, Anti-Oxidant Properties and Uses

Abstract

Black garlic is a fermented product obtained by aging fresh garlic under controlled temperature and humidity conditions, typically around 70°C and 90% relative humidity. This process enhances its taste, reduces its pungent odour, and significantly boosts its nutritional and medicinal value. The transformation of unstable sulphur compounds into stable, water-soluble antioxidants like S-allyl cysteine (SAC) increases its therapeutic potential. In this study, black garlic was further fermented using selected lactic acid bacteria (LAB) strains such as Lactobacillus plantarum and Lactobacillus rhamnosus to enhance bioactive compound content. The impact of this fermentation on antioxidant levels, flavour, and functional properties was evaluated. Black garlic extract showed increased levels of polyphenols, flavonoids, and other beneficial metabolites compared to fresh garlic. Antioxidant activity was measured using DPPH, ABTS, and FRAP assays, all confirming significant free radical scavenging capacity. Bioactive compounds such as SAC, 5-HMF, and organosulfur derivatives were found to contribute to anti-inflammatory, anticancer, and antimicrobial properties. The improved nutrient bioavailability and sensory characteristics make black garlic a promising nutraceutical. It may support cardiovascular health, blood sugar control, and oxidative stress reduction. Its high antioxidant content also suggests applications in preventing age-related diseases. This study contributes to understanding how fermentation and processing affect black garlic’s health-promoting potential. The results are relevant for both scientific research and the development of functional organic foods.

Keywords

Introduction

Garlic has been one of the most important foods and spices for centuries, widely used not only for its strong flavor in cooking but also for health benefits used in traditional medicine [1]. Garlic has been studied a lot and it’s known to be good for your body and health. In laboratory experiments (in vitro) and in live animals (in vivo), it has shown properties like killing harmful microbes and helping to stop the growth of cancer cells [2]. For a very long, time people have used plants to treat illness and stay healthy. In the last 20 years, more people around the world have started paying attention to traditional medicines, in both developing countries and developed countries. According to the world health organization up to 65% of people in developed countries use traditional medicine and treatments made from natural ingredients to stay health or treat illnesses[3].All over the world, functional and nutraceutical foods are becoming more popular because many people believe these foods can improve their health, boost their immunity and helps to prevent various diseases, easy to find and affordable for many people[4].Which can protect and maintain the health body. The improved therapeutic effects of black garlic are due to the presence of several active ingredients[5].Phenolic compounds play an important role in black garlic because they help provide health benefits, and they are also used to check the quality of fresh garlic products[6].Aged black garlic is made by storing whole garlic bulbs at a high temperature  of about 70°C  and in very humid conditions (around 90% humidity) for several days or weeks, which changes its colour, taste, and health properties[7].This study aims to find out what changes or health benefits happen when black garlic is fermented using helpful bacteria called LAB(lactic acid bacteria),first, the growth ability of three LAB strains -Lacto plant bacillus plantarum X7021,X7022, and Limosi  lactobacillus fermentum S1L23 was tested to see how well they could grow[8].In this study, we aimed to explore how fermenting black garlic extract with two types of lactobacillus bacteria -L plantarum and Lactobacillus rhamnoses. Affects its overall quality, especially focusing on changes in taste, smell (flavour) and beneficial compounds that support health [9]. It has a strong, spicy, and sometimes irritating taste, which makes it hard to use often as a food ingredient. this strong flavour comes from a natural compound called allicin and other similar substances (Jang et al.,2018). Alysin is one of the main substance found in garlic [10]. Because garlic can smell too strong for many people, researchers have tried several ways in recent years to make it taste and smell milder, heating the cloves -described by wang, Zhang and Jing (2016) and letting them age or ferment reported by Kim et al (2016) are the main techniques. These methods get rid of the strong smell and make garlic taste better and easier to eat[11].

Fig1: BLACK GARLIC [12]

The milder reaction, which occur between reducing sugars and proteins peptides or amino acids during food processing or storage, can produce various production with antioxidant activity (Del Castillo et al.,2002) Delgado Andrade et al., 2005; forage et al ;1982; friedman,1996; Nicoli et al., 1997; somazo2005 [13]. The concentration of s-allyl cysteine (sac)in black (BG) cloves is about two times higher than that found in black garlic extract indicating that the solid form retains more bioactive compounds. Compounds to aged garlic extract (AGE) black garlic after the benefit of a shorter manufacturing process, which enable the faster production of valuable functional compounds. In addition, since aged garlic extract is a liquid extract the ethanol used as a solvent must be removed before composition in contract black garlic can be consumed directly without further processing [14]. In the first case research focuses on the antioxidants precent in the Garlic as they may play a role in protecting the human body from the oxidative damage. This damage is caused by reactive oxygen species (ROS), which are unstable molecules that can harm cells and contribute to again and diseases [15]. Garlic contains, sulphur compounds like allicin, diallyl trisulfide which are through to be mainly responsible for its health benefits (Ankri and mirelman 1999; kumar and Berwal,1998) [16]. In studies of shallot extract have found flavonoids and polyphenols like quercetin, quercetin 4-glucoside, quercetin 7,4 di glucoside, quercetin 3,4-diglucoside and quercetin mono -d-glucose, which suggests that shallots may also have present in antioxidant properties [17]. But no one has tried using CaCl? soaking and freezing together to make black garlic before. Also, no one has studied the amounts and types of compounds like GSAC, SAC, and 5-HMF in black garlic made this way. [18]. This results of this study can help organic and healthy food procedure, as well as consumers who are looking for foods that are rich in nutrient and good for health [19]. And now currently about 300 types fresh garlic are known and 90 of them are registered in the European union. The most commonly used types are allium sativum which means cultivated garlic and allium ursine also known as bears garlic (Hanen et at;2012; Mayer et al; 2003) [20].  this, scientists also compared different types also compared to see how well they work as antioxidants.

Fig 2: VISUAL CHANGES DURING AGING OF GARLIC[21]

Fig 3: BIOLOGICAL ACTIVITIES OF BLACK GARLIC [24]

When garlic was processed at 60°c and 90% relative humidity for 45 days, its polyphenol content measured as g(GAE/kg) increased by 2.8%, and its antioxidant activity (measured as TROLOX equivalent kg) increased by 6.7% is ccompare to fresh garlic, black garlic has 3 to4 times more phenol-like compounds, 1.5 to 8 times more flavonoid-like compounds, and a 1.6 to 12 times more antioxidant compounds. Overall, black garlic has a lot more polyphenols than fresh garlic—about three times more in whole bulbs and up to six times more in peeled cloves. [25]. Black garlic also contains cysteine, a sulphur-containing amino acid that helps build proteins and supports antioxidant activity in the body. The aging process of garlic increases the amount of beneficial polyphenols in black garlic which also boosts its antioxidant power. During the aging process of garlics unstable and strong - smelling compounds are changed into S-allyl cysteine which helps increases an antioxidant power of black garlic. S-allyl cysteine is a strong antioxidant that mixes easily with water. A Eexperimental results showed that the amount of S-allyl cysteine increased sixfold compared to raw garlic which contains 20–30 µg/g [26]. The composition includes steroidal glycosides, essential oils, polyphenols, and a variety of vitamins such as B1, B2, B6, C and E (Bozin, Mimica-Dukic, Samojlik, Goran, & Igic, 2008; Samarth, Samarth [27].

Fig 4: Comparison of the components in black garlic and fresh garlic.[28]

Fig 5: PREPARATION AND FERMENTATION PROCESS OF FRESH GARLIC AND BLACK GARLIC[32]

Fig 6: HEALTH BENEFITS OF BIOACTIVE COMPOUNDS[35]

Fig 7: BIOACCESSIBILITY OF BIOACTIVE COMPOUNDS FROM FRESH GARLIC AND BLACK GARLIC[37]

EXTRACTION OF ANTIOXIDANTS

Black garlic is produced by fermenting whole garlic bulbs in a warm, humid environment, which transforms their flavour and boosts their nutritional value. This process boosts its antioxidant levels, making it more beneficial for health than fresh garlic. The antioxidant compounds, like polyphenols and flavonoids, are extracted through various methods, including maceration with solvents like ethanol or ethyl acetate, or hot water extraction. These extracts are then often analysed for antioxidant activity using techniques like DPPH, ABTS, and FRAP assays [2].

Maceration:

Black garlic is soaked in a solvent (often ethanol) for a period of time, allowing the soluble compounds to dissolve.

Solvents:

Common solvents include ethanol (e.g., 70% ethanol), ethyl acetate, and n-hexane [3]

Fractionation:

Techniques like TLC or flash chromatography are used to separate different compounds from the extract based on their properties.

ANTIOXIDANT EXTRACTION PROCESS

DPPH technique

The DPPH method is a common spectrophotometric assay used to determine the antioxidant activity of black garlic extracts. It involves reacting a DPPH solution with the black garlic extract and measuring the decrease in absorbance caused by the neutralization of free radicals. This method helps quantify the extract's ability to scavenge free radicals, a key indicator of antioxidant capacity. [38]. Here's a more detailed breakdown of the process:

1. Preparation of Black Garlic Extract:

• Black garlic is prepared by aging fresh garlic under controlled temperature and humidity, resulting in increased antioxidant compounds[2].
• The black garlic is then extracted using various methods, such as maceration with ethanol or other solvents.
• The extract is often concentrated using a rotary evaporator and may be further purified into different fractions[3].

2. Preparation of DPPH Solution:

• A solution of DPPH (2,2-diphenyl-1-picrylhydrazyl), a stable free radical, is prepared using methanol as a solvent.
• The DPPH solution is typically a 100ppm solution.
• The maximum absorbance wavelength of the DPPH solution is determined using a UV-Vis spectrophotometer.[39]

3. DPPH Assay:

• Various concentrations of the black garlic extract are prepared.
• The DPPH solution is added to the extract solutions and allowed to react for a specific time (e.g., 30 minutes) in the dark.
• During the reaction, the antioxidants in the extract scavenged the DPPH free radicals, resulting in a colour change from purple to yellow.
• Solution absorbance was recorded using a UV-Vis spectrophotometer set to the specified wavelength [40]

4. Data Analysis:

• The absorbance values are used to calculate the percentage of DPPH radicals scavenged by the extract.
• The  IC?? value was determined as the concentration of the extract required to inhibit 50% of DPPH radical activity.
• Lower IC50 values indicate higher antioxidant activity [41].

 EXTRACTION

• Solvent Selection: Ethanol is commonly used for extracting antioxidants from black garlic.
• Extraction Method: Soxhlet extraction or maceration are suitable methods for extracting the compounds.
• Soxhlet extraction:  finely chopped black garlic was placed in a thimble and repeatedly extracted using 96% ethanol until the solvent in the siphon tube turned clear.
• Maceration:  Black garlic mixed with 70% ethanol solvent (1:10 w/v) in a maceration bottle, stirring occasionally, and soaked for 6 hours before standing for another 18 hours. The extraction procedure was repeated using the same solvent type and volume. The resulting extract was filtered to remove particulate matter and subsequently concentrated to a viscous consistency using a rotary evaporator maintained at a controlled temperature below 40–50?°C.
• Evaporation: Use a rotary evaporator at a moderate temperature (e.g., 40°C) to obtain a concentrated extract.
• Determine the extract's yield and characterize it (e.g., organoleptic properties, drying shrinkage, ash content, phytochemical screening) [3].

ANTI OXIDANTS EXTRACTION BY ABTS METHOD

The extraction of antioxidants from black garlic using the ABTS technique involves preparing a black garlic extract, generating the ABTS radical cation, and then measuring the extract's ability to scavenge this radical, indicating its antioxidant capacity. The process typically includes preparing the black garlic, extracting the bioactive compounds, and then using the ABTS assay to quantify antioxidant activity [42].

1. Black Garlic Preparation and Extraction:

• Black garlic is prepared by aging fresh garlic at high temperature and humidity, resulting in the formation of various bioactive compounds like S-allyl cysteine (SAC) and other antioxidant [43]
• The black garlic is then peeled and ground into a powder or paste [44].
• A solvent, often water or a water-ethanol mixture, is used to extract the antioxidants from the black garlic powder [45].
• The mixture is typically heated and or sonicated to enhance extraction efficiency [46].
• The extract was subsequently filtered to eliminate any residual solid particles[47].

2. ABTS Radical Generation and Assay:

• The ABTS? radical cation is formed by reacting ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) with a strong oxidizing agent such as potassium persulfate.
• This generates a stable radical that has a characteristic absorbance at a specific wavelength.
• Black garlic extract was mixed with ABTS? solution to evaluate its antioxidant activity.
• The decrease in absorbance of the ABTS + solution after the addition of the extract indicates the extract's ability to scavenge the radical.
• The antioxidant activity is usually expressed as a percentage of ABTS radical scavenging or as an EC50 value (the concentration of the extract needed to scavenge 50% of the ABTS radicals) [48].

3. Analysis and Interpretation:

• To assess antioxidant activity, the absorbance of the ABTS? solution was recorded using a UV-Vis spectrophotometer at 734?nm, which is the standard wavelength for this assay. [49].
• The percentage of ABTS radical scavenging is calculated using the formula: ABTS + radical scavenging effect (%) = [1 - (absorbance sample / absorbance control)] × 100[50]
• The results are then compared with a standard antioxidant such as Trolox, to measure how strong the antioxidant activity of the black garlic extract [1].
• The ABTS assay is a well-established and reliable method for evaluating the antioxidant capacity of various compounds, including black garlic extract. [51].

ANTIOXIDANT EXTRACTION BY USING THE FRAP ASSAY

The FRAP (Ferric Reducing Antioxidant Power) assay is a method used to determine the antioxidant capacity of black garlic extracts by measuring their ability to reduce ferric ions to ferrous ions. This reduction is evidenced by a colour change, the intensity of which correlates with the antioxidant activity. Black garlic, produced by aging fresh garlic under controlled conditions, exhibits higher antioxidant activity compared to fresh garlic, largely due to the formation of compounds like S-allyl cysteine and tetrahydro-β-carbolines during the aging process [52].

HERES A BREAKDOWN OF THE PROCESS:

1. Preparation of Black Garlic Extract:

• This process transforms the garlic’s flavour, texture, and nutritional profile—enhancing its antioxidant content and reducing its pungency, making it sweeter and more palatable [1].
• The black garlic is then extracted using a suitable solvent, often methanol or water, to obtain a solution containing antioxidant compounds [53]. 
• The extract may be filtered and concentrated before use in the FRAP assay [54].

FRAP Reagent Preparation:

The FRAP reagent was freshly prepared by combining acetate buffer with TPTZ (2,4,6-tripyridyl-s-triazine), and FeCl3 (ferric chloride). [55]

2. FRAP Assay Procedure:

• The FRAP reagent is usually prepared using a ferric-tripyridyl triazine (Fe³?-TPTZ) complex, which serves as the oxidizing agent in the assay.
• The diluted black garlic extract is combined with the FRAP reagent to assess its antioxidant capacity. [56]
• The mixture is incubated at a specific temperature (e.g., 37°C) for a set time. 
• The absorbance of the reaction mixture is measured at a specific wavelength (e.g., 593 nm) [57].

3. Interpretation of Results:

• The absorbance value is compared against a standard curve created using known concentrations of a reference antioxidant, such as Trolox or ascorbic acid, to quantify the antioxidant capacity of the sample.
• The FRAP value, expressed as mmol/L or mg/L of antioxidant equivalent, indicates the antioxidant capacity of the black garlic extract.
• A higher FRAP (Ferric Reducing Antioxidant Power) value reflects stronger reducing ability and greater antioxidant activity in the sample [58].

4. Black Garlic and FRAP:

• Black garlic has been shown to exhibit higher antioxidant activity compared to raw garlic, as measured by FRAP assays. 
• This increased antioxidant capacity is often attributed to the formation of various bioactive compounds during the aging process, such as S-allyl cysteine, tetrahydro-β-carbolines, and melanoidins. 
• The FRAP (Ferric Reducing Antioxidant Power) assay is a useful method for measuring the antioxidant potential of black garlic, helping to reveal its health-promoting properties. [59].
• In essence, the FRAP assay provides a quantitative measure of the reducing power of black garlic extracts, reflecting their potential to scavenge free radicals and protect against oxidative stress [60].

WHY FRAP ASSAY IS USED FOR BLACK GARLIC

Simple and Reproducible:

The FRAP assay is relatively simple to perform and provides reproducible results, making it a reliable method for comparing antioxidant activity across different black garlic samples.

Measures Reducing Power:

It directly measures the reducing power of antioxidants, which is a key aspect of their antioxidant activity.

Widely Used:

The FRAP assay is a widely accepted method for assessing antioxidant capacity, making it easy to compare results with other studies [61].

Key points about antioxidants in black garlic:

• Black garlic contains a variety of antioxidant compounds, including S-allyl cysteine, tetrahydro-β-carbolines, flavonoids, and melanoidins. 
• During the aging process, black garlic exhibits increased antioxidant activity due to the generation of these compounds. 

The FRAP assay is a valuable tool for quantifying the overall antioxidant capacity of black garlic extracts and understanding the impact of different processing methods on their antioxidant potential [62].

CONCLUSION

Black garlic is a fermented type of garlic that may be good for health because it has better nutrients and stronger effects than fresh garlic. Studies show it has antioxidant, anti-inflammatory, and anticancer properties, but more research is needed to fully understand how it works and confirm its benefits.

Improved Nutrient Profile:

Black garlic has more antioxidants, like polyphenols and flavonoids, and also contains special compounds such as S-allyl cysteine.[63]

Enhanced Bioavailability:

Fermentation helps increase the availability of certain compounds in black garlic, so the body can absorb them more easily and use them better.[64]

Studies show that black garlic might be helpful in areas such as:

It may help protect the body from cell damage and reduce inflammation.

Cardiovascular Health:

These effects are linked to its rich antioxidant content, which supports heart health and may reduce the risk of cardiovascular disease.

Antioxidant and Anti-inflammatory Activity:

Its antioxidants might improve the function of blood vessels and reduce inflammation.

Cancer Prevention:

 Some studies show that black garlic might help stop cancer cells from growing.

Blood Sugar Regulation:

 Black garlic may help control blood sugar, which could be useful for people with diabetes [65].

Cardiovascular Health and Cholesterol Management:

Consuming black garlic has been linked to improved cholesterol profiles, including a reduction in LDL (bad) cholesterol and an increase in HDL (good) cholesterol. Such improvements in lipid profiles and blood pressure regulation contribute significantly to cardiovascular health and may reduce the risk of heart disease.

Blood Pressure Regulation:

Black garlic may help reduce blood pressure by promoting vasodilation and minimizing oxidative stress, both of which contribute to healthier vascular function.[66]

Improved Blood Circulation:

Black garlic may increase nitric oxide levels, which helps improve blood flow and circulation in the body.[67]

Blood Sugar Control (or) Regulation of Blood Sugar:

Black garlic may help control blood sugar, which could be useful for managing diabetes and its complications.[68]

Antibacterial Activity:

Black garlic has been found to fight certain bacteria like E. coli, which means it might help treat bacterial infections.

Anticancer Potential:

Research shows that black garlic may slow the growth of tumours and stop cancer cells from spreading, possibly because of its antioxidants and other active compounds.

Other Potential Benefits:

Black garlic may also help protect the liver and brain, and it might help reduce obesity.[69]

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