Black garlic: A critical review of its production, bioactivity, and application

2.1. Comparison of the components between fresh garlic and BG

Fresh garlic contains approximately 63% of water, 28% of carbohydrate (fructans), 2.3% of organosulfur compounds, 2% of proteins (alliinase), 1.2% of free amino acids(arginine), and 1.5% of fiber [9]. Nontreated fresh garlic also contains a high amount of γ-glutamylcysteines [10]. These compounds can be hydrolyzed and oxidized to form alliin, which accumulates naturally during the storage of garlic at a cool temperature. After processing, such as cutting, crushing, chewing, or dehydration, alliinase rapidly lyses the cytotoxic cysteine sulfoxides (alliin) to form cytotoxic and odoriferous alkyl alkane-thiosulfinates such as allicin [11]. Allicin contributes to the characteristic flavor and taste of garlic. Allicin and other thiosulfinates are immediately decomposed to other compounds such as diallyl sulfide, diallyl disulfide, and diallyl trisulfide, dithiins, and ajoene [11], [12]. At the same time, γ-glutamylcysteines are converted to SAC through its catabolism pathway other than the alliin–allicin pathway [13]. SAC contributes to health benefits of garlic, such as its antidiabetic, antioxidant, and anti-inflammatory activities [14], [15], [16].

As for BG, during the thermal process, some chemical compounds from fresh garlic are converted into Amadori/Heyns compounds, which are key intermediate compounds of Maillard reaction [1]. The chemical compounds of aged BG (ABG) are complicated, and the quality of its products depends on the manufacturing process. Nevertheless, BG contains much more functional compounds such as SAC than fresh garlic.

The contents of chemical compounds of BG depend on the conditions during thermal processing. Some researchers reported that many valuable components within BG against diseases increased during the aging process, especially polyphenol, flavonoids, and some intermediates of Maillard reaction have been known as antioxidant agents [13], [17]. Furthermore, the antioxidant activity of garlic varies across regions [18]; nevertheless, BG demonstrates significantly much higher biological activity, such as antioxidant properties, than fresh garlic [19].

3.1. Effects of aging temperature on the quality of BG

It is well known that the aging period of garlic is shorter at higher temperatures [22]. In the case of aging process at 70°C, the speed of aging is two-fold faster than that at 60°C [23]. According to sensory evaluation, the quality of BG is better and its black color is homogeneous between 70°C and 80°C [23]. Even though BG is produced faster at 90°C, it produces nonideal tastes, such as bitter and sour tastes [23]. In the case of aging process at 60°C, the color of garlic was not completely black; thus, 60°C is also not an ideal condition for the aging process.

When the moisture content of garlic reaches 400–500 g/kg, BG can be suitable for eating because of its softness and elasticity. If moisture content is about 350–400 g/kg, BG would be much drier and its elasticity would be poor. In particular, when moisture content goes below 350 g/kg, BG becomes too hard to eat [23]. Moreover, the aging speed of fresh garlic to BG is markedly slow when processed at 60°C. Although aging occurs smoothly at 80°C and 90°C, an adequate condition is relatively difficult to find because of its fluctuating phenol content and reducing sugar content [23].

Content of reducing sugar is also considered an important factor during the aging process. Some types of sugar and amino acids are required for Maillard reaction [24]. The reducing sugar content gradually increases at 60°C and 70°C during the whole process, which means that at these temperatures, the rate of formation of reducing sugar is faster than its rate of consumption. Although the content of reducing sugar increases at high temperatures, in the case of processing at 80°C and 90°C, ABG does not have an appropriate sweet flavor because of the consumption of a large amount of reducing sugar at high-temperature conditions [23]. Besides, reduction of amino acid content is also accelerated depending on the progress of Maillard reaction [23].

One of the main antioxidant compounds in BG is 5-hydroxymethylfurfural (5-HMF), and it is also an important intermediate product in Maillard reaction [23]. Regardless of temperature, the amount of 5-HMF is increased during the aging process. However, in the case of processing at 60°C, 5-HMF content increases very slowly during the whole process.

3.2. Effects of fermentation condition on the quality of BG

As we mentioned in the previous section, the quality of BG including its bioactivity and texture depends on the temperature during thermal processing. However, according to the discovery of Jung et al [25], fermented BG displays more effective bioactivity than ABG. In this section, we will summarize the quality of fermented BG and its potentials against several kinds of diseases.

Improvement of antioxidant activity will effectively prevent diabetes and its related complications [26], [27]. Bioactivities of garlic such as antioxidant activity and hypoglycemic effect are already well known, and the antioxidant activity of garlic could be enhanced by processing. In recent years, Hien-Trung et al [28] discovered that the bioactivity of ginseng could be enhanced by yeast fermentation. Therefore, they hypothesized that the bioactivity of BG may also be enhanced by yeast fermentation.

According to Jung et al [25], yeast-fermented BG exhibited much better bioactivity against syndromes such as obesity, hyperlipidemia, nephropathy, and hepatopathy than ABG. For example, yeast-fermented garlic-treated mice demonstrated marked improvement in body weight, periovarian fat weight, adipocyte diameters, deposited abdominal fat pad thicknesses, serum total cholesterol, triglyceride, low-density lipoprotein (LDL) level, high-density lipoprotein (HDL) level, aspartate transaminase (AST), alanine transaminase (ALT), steatohepatitis, hepatocyte hypertrophy, serum blood urea nitrogen (BUN), and the number of abnormal kidney tubules compared with the high-fat diet (HFD)-treated controls. Furthermore, fermented BG 400 mg/kg and 200 mg/kg revealed significantly higher effects than ABG 400 mg/kg. In other words, fermented BG has more effective bioactivity against HFD-induced obesity, hyperlipidemia, nephropathy, and hepatopathy than ABG [25]. Therefore, the bioactivity of BG could be enhanced by yeast fermentation, and fermented BG may be more qualified to improve diabetes and its related complications. Owing to this reason, the components of fermented BG might be more or less different from those of ABG. However, the differences of the components between ABG and fermented BG have still not been investigated. Therefore, their component analysis should be required.

In summary, the aging period of BG is shorter at a high temperature; however, controlling the amount of some components might be difficult at a high temperature because their contents change rapidly during the aging process. Based on the results mentioned above, 70°C is considered the best condition for garlic aging. However, the quality of BG is affected by not only temperature, but also other factors such as humidity and fermentation [23], [25]. Therefore, further investigations are also required.

4.1. Antioxidant activity

The antioxidant activity of garlic is affected by the ways of processing [42]. Alliin is an unstable compound in fresh garlic, which is converted into a stable compound, SAC, during the aging process and exhibits antioxidant activity [11], [31], [32]. Lee et al [32]reported that the decrease in the number of free radicals in ABG (59.2 ± 0.8 μmol/g wet weight) is more than that in garlic (13.3 ± 0.5 μmol/g wet weight), as revealed by the trolox equivalent antioxidant capacity (TEAC) assay in vitro[32]. Another study showed that 10 μg/mL of yeast-fermented BG had stronger antioxidant activity than BG, as detected by the in vitro electron-donating ability assay [25]. Fresh garlic undergoes 40 days of fermentation at 60–70°C and 85–95% relative humidity to produce BG. The BG extract had more than 10-fold increase in superoxide dismutase-like activity and scavenging activity against hydrogen peroxide compared with garlic extract in vitro[19]. Kim et al [34] showed that the formulation containing 10% of BG extract had higher radical scavenging activity than the formulation containing 10% (v/v) of garlic extract by 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) assays in vitro. ABG was obtained from fresh garlic fermented at 80–90°C for 48–90 hours, at 70–80°C for another 48–60 hours, then at 60–70°C for 72–120 hours, and finally at 55–65°C for 72–120 hours. ABG also showed stronger antioxidant activity than fresh garlic by DPPH and 2,2-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) assays [3]. Seventy percent ethanol extract of BG had higher DPPH radical scavenging activity than 70% and 90% ethanol extracts of raw garlic and 90% ethanol extract of BG [35].

4.2. Inhibition of cancer cell line growth

There are six characteristics of cancer during the multistep development of human tumors including sustained proliferative signaling, evaded growth suppressors, resisted cell death, enabled replicative immortality, induced angiogenesis, and activated invasion and metastasis. Therefore, functional food could block these six characteristics due to their anticancer ability [43].

The hexane extract of ABG (HEABG) had demonstrated its anticancer activity in human leukemic U937 cells. HEABG (2.5 μg/mL, 5 μg/mL, 7 μg/mL, and 10 μg/mL) inhibited cell growth by inducing the intrinsic pathway of apoptosis via upregulation of death receptor 4 and Fas ligand, and increasing the Bax/Bcl-2 protein expression ratio. HEABG also activated caspase-9 and caspase-3, and degraded poly(ADP-ribose)-polymerase in a concentration- and time-dependent manner. HEABG-inhibited cell growth also induced the extrinsic pathway of apoptosis via activated caspase-8, resulting in the truncated Bid expressed. HEABG showed its potential for anticancer ability by inducing caspase-dependent apoptosis through both intrinsic and extrinsic pathways in human leukemic U937 cells [4].

Another study showed that 70% ethanol extract of BG (500 μg/mL) caused cytotoxicityin human carcinoma A549 (lung carcinoma), MCF-7 (breast adenocarcinoma), AGS (stomach adenocarcinoma), and HepG2 (hepatocarcinoma) cells in a dose-dependentmanner within 72 hours [35].

4.3. Antiobesity activity

Obesity is an inducer of other diseases such as type 2 diabetes, heart disease, liver disease, and the phenomena of liver damage, including hyperlipidemia[44], changes in liver weight, and serum AST and ALT levels [45].

Female Institute for Cancer Research (ICR) mice were fed with 45%/kcal of HFD for 28 days and then the mice were given 400 mg/kg BG and 100 mg/kg, 200 mg/kg, and 400 mg/kg of yeast-fermented BG for 63 days. BG and yeast-fermented BG significantly decreased body weight, abdominal fat weight, abdominal adipocyte diameters, and abdominal fat pad thickness compared with the HFD group. BG and yeast-fermented BG also decreased serum triacylglyceride and LDL levels, and increased serum HDL level compared with the HFD group. BG and yeast-fermented BG decreased serum AST, ALT, steatohepatitis, and hepatocyte diameters compared with the HFD group [25]. Male Sprague–Dawley rats were divided into four groups, and fed with normal food diet, HFD, and HFD + 0.5% or 1.5% BG extract for 5 weeks. The results showed that rats from the 1.5% BG extract group had decreased weight gain and epididymal fat compared with the HFD group. BG extract [1.5% (w/v)] also showed decreased triacylglyceride in the serum and liver and an increased HDL level in the serum [5].

4.4. Hepatoprotective function

Male Sprague–Dawley rats were fed with ethanol to induce oxidative liver damage. The mice were also given 100 mg/kg of ABG by oral gavage. The results showed that ABG decreased body weight and total fat pad weight. The plasma markers of liver function and injury, including AST, ALT, ALP, and LDH levels, were significantly decreased by ABG compared with those in the group treated with ethanol alone. ABG also increased CYP2E1 expression and the activities of glutathione S-transferase and quinone reductase were drug-metabolizing phase II enzymes and restored the thiobarbituric acid reactive substances, glutathione level, the activities of glutathione peroxidase, GR, and catalase in the liver [38].

Another study showed that 200 mg/kg ABG decreased ALT and AST levels in the liver in the carbon tetrachloride- and d-galactosamine-induced liver damage models of Sprague–Dawley rats. It also decreased the ALT and AST levels in HFD-induced fatty liver and subsequent liver damage model of C57BL/6 mice [39].

4.5. Immunomodulatory effect

Atherosclerosis is a chronic inflammatory disease of the arterial walls due to endothelial dysfunction, vascular inflammation, and formation of atheromatous plaque within the intima of the vessel wall. Atherosclerosis is also related to increased oxidative stress caused by vascular inflammation with various cytokines, including tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, and interferon-γ, inducing endothelial activation via generation of reactive oxygen species and augmenting the expression of cell adhesion molecules on endothelial cells [41], [46].

Previous researches showed that BG had antioxidant ability [31], [32]. Dr Yoon’s group had been investigating the effect of different extraction methods of ABG in the TNF-α-stimulated human umbilical vein endothelial cell (HUVEC) model. Chloroformextract (30 μg/mL) of ABG was pretreated in TNF-α-stimulated HUVECs. This ABG extract inhibited reactive oxygen species formation and mRNA expression of vascular cell adhesion molecule-1 (VCAM-1), and reduced THP-1 monocyte adhesion to TNF-α-stimulated HUVECs. Chloroform extract of ABG also inhibited the activation of nuclear factor kappa B (NF-κB) transcription factor in TNF-α-stimulated HUVECs [40].

The compound 5-HMF was found in chloroform extract of ABG and treated in TNF-α-stimulated HUVECs. It suppressed total protein and mRNA expression of VCAM-1 and intercellular cell adhesion molecule-1 (ICAM-1) in TNF-α-induced cell surface. It also inhibited reactive oxygen species formation, THP-1 monocyte adhesion, and activation of NF-κB transcriptional factor in TNF-α-stimulated HUVECs [41]. HEABG (50 μg/mL) suppressed cell proliferation and cell cycle progress via extracellular signal–regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) pathways in TNF-α-activated human endometrial stromal cells isolated from patients with endometriosis. HEABG also had the potential to suppress the TNF-α-induced ICAM-1 and VCAM-1 transcripts and protein expression via inhibition of the activation of NF-κB and AP-1 transcription factors [47].

Lipopolysaccharide (LPS) is an endotoxin that induces several cytokines, such as TNF-α, IL-1b, and IL-6, which are related to various inflammatory reactions [48]. There is another research in which the MTT assay showed that raw garlic extract was highly cytotoxic at concentrations over 250 μg/mL with or without LPS in the RAW 264.7 cells. Water extract of ABG (WEAGE) did not show significant cytotoxicity up to 2000 μg/mL. The result showed that WEAGE had less cytotoxicity than raw garlic extract. When WEAGE was added again at 15 hours before LPS was added to the cells, the results showed that WEAGE decreased the production of nitric oxide (NO), TNF-α, and prostaglandin-E2 in a dose-dependent manner in LPS-stimulated RAW 264.7 macrophages via downregulation of NO synthase and TNF-α mRNA expression, and cyclooxygenase-2 protein expression. Moreover, its anti-inflammatory mechanism decreased LPS-induced phosphorylation of JNK and p38MAPK, and inhibited the activation of NF-κB and phosphorylation in response to LPS-stimulated RAW 264.7 cells. The authors fed the C57BL/6 mice 120 mg/kg of WEAGE and raw garlic extract by oral gavage before injecting 20 mg/kg LPS (LPS-induced endotoxemia). WEAGE decreased the level of TNF-α and IL-6 in the serum against LPS-induced lethal shock in C57BL/6 mice [6].

4.6. Antiallergic action

More evidence showed that allergy diseases are influenced by environmental factors such as eating habits, stress, and living environment. In fact, number of allergy patients have been increasing in many countries [49].

Allergy is related to immunoglobulin E (IgE) antibodies and mast cells have to respond to the pathophysiology of anaphylaxis and other acute allergic reactions. Lots of evidence shows that IgE and mast cells have key roles in tissue remodeling that is associated with chronic allergic inflammation in asthma. Allergy is classified into five types. At type I allergy responses as anaphylactic type can be activated by the high-affinity IgE receptor (FcɛRI receptor) on the plasma membrane of mast and basophilic cells as an intragranular mediators such as histamine, arachidonic acid metabolites, proteases, serotonin, and heparin and it can release β-hexosaminidase, a general marker of degranulation. Therefore, mast cells have an important role in allergic reactions [50], [51]. RBL-2H3 cells are used as a model for screening allergic reactions in vitro and passive cutaneous anaphylaxis as an animal model for screening IgE-mediated allergic responses [52], [53]. Ethyl acetate extract of BG (2 mg/mL) inhibited the release of β-hexosaminidase and TNF-α that inhibited IgE-mediated allergic responses in RBL-2H3 cells. Moreover, BG10 was the active fraction from ethyl acetate extract of BG showing stronger inhibition of the release of β-hexosaminidase and TNF-α compared with other fractions. Furthermore, 50 μg/mL of BG10 inhibited the formation of prostaglandin E2 and leukotriene B4, and phosphorylation of Syk. BG10 also decreased the phosphorylation of cytosolic phospholipase A2 and 5-lipoxygenase, and the expression of cyclooxygenase-2 in RBL-2H3 cells. BG10 (66.7 mg/kg) given to mice by oral gavage for 1 hour decreased the passive cutaneous anaphylaxis reaction on IgE-mediated passive cutaneous anaphylaxis reaction in mice [7].

4.7. Reduction of blood lipid

Previous studies showed that BG improved serum lipid profiles such as total cholesterol, triglycerides, LDL, and HDL with mice fed with HFD [25].

Jung et al [54] showed that ABG could improve blood lipid profiles in patients with mild hypercholesterolemia. Sixty participants were divided into two groups. One was given 6 g ABG and the other was given placebo twice per day before a meal every morning and evening for 12 weeks. Although the ABG group did not show significant differences in triglyceride, LDL cholesterol, total cholesterol, or free fatty acid levels compared with the placebo group, ABG increased HDL cholesterol levels compared with the placebo group at the end of the study [54]. Serum apo B (atherogenic lipoprotein) is an independent and high predictive risk factor for coronary artery disease [55]. In conclusion, ABG supplement significantly decreased serum apo B [54].

4.8. Influences on memory and nervous systems

Monosodium glutamate (MSG) is well known, and has been used for seasoning all over the world due to its attractive umami taste [56]. However, some researchers reported that MSG might have adverse effect on various organs including Purkinje cells in the cerebellum and hippocampus [57], [58]. The cerebellum and the hippocampus play an important role in the nervous system and the memory system, respectively. As it is, the brain is expected to be one of the organs most sensitive to the effects of MSG because of its high content of polyunsaturated fatty acids, high metabolism, and low antioxidant capacity, and the hard-to-replicate quality of its neuronal cells [59], [60], [61].

+Garlic has been known not only as a flavor enhancer, but also as a food that has high potential antioxidant activity. In particular, the antioxidant activity of BG is significantly higher (p < 0.05) than that of fresh garlic due to its higher polyphenol level and scavenging activity [56], [62]. Some researchers investigated the effects of the ethanol extract of BG on the nervous and memory systems using a Wistar rat model with MSG [63], [64]. According to Hermawati et al [63], BG-treated rats had significantly shorter escape latencies and path lengths than the control rats, both with or without MSG, in several trials of the nonvisible platform test of the Morris Water Maze (MWM) procedure. Furthermore, although the dosage of MSG may not be adequate to show significant reduction of the number of Purkinje cells, the combined administration of BG extract and MSG improved the reduction of the number of Purkinje cells compared with MSG only [64].

To sum up, BG might play an important role in improving some diseases as well as the functions of the memory and nervous systems due to its potential antioxidant activity. However, the current dosage of MSG may not be able to significantly reduce the number of Purkinje cells in the cerebella of rats. Therefore, further studies are required to discover whether a higher dosage of MSG can affect the number of Purkinje cells.

4.9. Influence of BG on TNF-α-related inflammation diseases

Accumulation of monocytes in the vessel wall is primarily induced by specific cell adhesion molecules such as VCAM-1, ICAM-1, and endothelial cell selection [40]. In particular, VCAM-1 is activated by cytokines such as TNF-α and IL-1 in the endothelium. This phenomenon could be attributed to atherosclerosis through the adhesion of monocytes in the endothelium. Furthermore, these cell adhesion molecules can result in endometriosis [42]. These are regulated by the expression of cytokines and chemokines.

Although TNF-α is well known as an inducer of expression of cell adhesion molecule, it is deeply involved in the inflammation reaction in humans. Following are the influences of BG extracts on TNF-α-related diseases.

Chloroform extract of BG could suppress cell adhesion molecules that are activated by TNF-α. Reactive oxygen species production, NF-κB activation, and adhesiveness to monocytes were also improved [40]. Moreover, 5-HMF, which is purified from BG, could also suppress cell adhesion molecules that are activated by TNF-α [41]. Furthermore, hexane extract of BG could reduce the expression of cell adhesion molecules such as ICAM-1 and VCAM-1 in TNF-α-activated endometrial stromal cells in humans.

In summary, hexane extract of BG could be effective in preventing and treating endometriosis in humans. Furthermore, chloroform extract of BG and 5-HMF could also be effective in preventing and treating atherosclerosis.However, the exact chemical compound which have bioactivity are still not investigated. Therefore, the compound analysis of them should be required.