What Is the Nutrition Fact of Black Garlic Extract?

Garlic (Allium sativum L.) belongs to the family Liliaceae and is known worldwide as a “natural antibiotic grown on land” that can be used as both a food and a cooking ingredient, as well as a traditional Chinese medicine. Garlic contains a variety of bioactive ingredients, which have a variety of effects, such as warming the spleen, promoting digestion and regulating qi, antibacterial and anti-inflammatory, and lowering blood sugar and blood lipids [1]. Garlic has a long history of cultivation in China, is widely planted, has a high yield, and has maintained a relatively high export volume for many years, ranking first in China's total exports of a single agricultural product. However, garlic has a special pungent odor and spicy taste, which can even cause gastrointestinal discomfort in some people, limiting its application. People have tried various processing techniques to treat garlic in order to minimize the garlic odor, improve its palatability, and maintain or enhance its beneficial biological functions.

Black garlic is made from fresh garlic that is processed under high heat and humidity for a certain period of time. Black garlic was first invented by the Japanese, and gradually developed and spread to the mainland through Korea, Spain, Taiwan, and other places. It is loved by consumers and has become a new favorite health food because of its good taste and strong physiological effects. Black garlic is soft, elastic, sweet and delicious, without the pungent taste and irritating odor of garlic. Compared with fresh garlic, the chemical composition of black garlic has undergone major changes, not only increasing the content of the original nutrients, but also producing some new functional substances, which have better physiological activity, such as anti-oxidation, regulating blood sugar and blood lipids, and anti-tumor.

This article mainly summarizes the research on the main nutritional components, biological functions, processing technology and endophytic bacteria of black garlic, and provides a perspective on the future development and application of black garlic.

1 Main nutritional components in black garlic

Garlic is very nutritious. Each 100 g of fresh garlic contains 63.8 g of water, 577.39 kJ of energy, 7.3 g of sugar, 5.2 g of protein, 0.2 g of fat, 10 mg of calcium, 12.5 mg of phosphorus, iron 1.3 mg, vitamin B1 0.29 mg, vitamin B2 0.06 mg, niacin 0.8 mg, vitamin C 7 mg. It also contains a variety of trace elements essential to the body. After garlic is processed through enzymatic, ripening, and drying processes to become black garlic, its nutritional content is greatly enhanced. The content of compounds such as saccharide compounds, organic acids, and total phenols increases, and new substances such as melanoidin and 5-hydroxymethylfurfural (5-HMF) are also produced, giving it higher nutritional value and physiological activity.

1. 1 Carbohydrates

Garlic polysaccharides are the main component of garlic, and have various physiological effects such as anticoagulation, lowering blood lipids, preventing atherosclerosis, anti-cancer and anti-tumor, lowering blood sugar and anti-aging. At present, related research focuses on the extraction, content determination and antioxidant properties of polysaccharides. The polysaccharide content of black garlic and garlic is 98.67 mg/g and 50.33 mg/g respectively [2]. Black garlic crude polysaccharides were obtained through a separation and purification process including hot water extraction, rotary evaporation concentration, centrifugation, protein removal by Sevag method, precipitation and washing, and drying. The extraction rate was 8.14%, and the purity of the crude polysaccharides was 43.0% as determined by the phenol-sulfuric acid method [3]. Papain was used to extract black garlic polysaccharides by the assisted water extraction and alcohol precipitation method, and the extraction method was optimized. The optimal extraction conditions were: enzyme dosage 1.5%, enzyme action pH 6.5, extraction temperature 55°C, ultrasonic time 75 min. Under these conditions, the extraction rate of black garlic polysaccharides can reach 10.15% [4]. Black garlic polysaccharides have good antioxidant properties and their health benefits deserve further research.

Compared to fresh garlic, black garlic becomes sweet and sour. During the processing, the enzymatic hydrolysis of amylase and fructanase and the heat during the maturing process gradually break down the polysaccharides in the garlic into monosaccharides (mainly glucose and fructose), disaccharides and oligosaccharides, which increases the sweetness of the black garlic. The finished black garlic contains more than 60% reducing sugars by dry weight, which is 30 to 80 times that of fresh garlic [5].

The sugar components of black garlic obtained by heat treatment mainly include fructose (57.14%), sucrose (7.62%), and glucose (6.78%). Among these, the increase in fructose is most significant [in black garlic (0.38 ± 0.06) (44.73 ± 4.41) g/100 g DM)], followed by glucose [(0. 21 ± 0. 02) ~ (2. 51 ± 0. 24 ) g/100 g DM ] [6 ]. During the processing of black garlic, the rate of accumulation of reducing sugars is related to the temperature. The faster the temperature increases, the faster the rate of accumulation of reducing sugars, which indicates that the degradation of garlic polysaccharides is mainly the result of high temperatures. However, when the effect of enzyme inactivation on the browning degree and reducing sugar content of black garlic was studied at a constant temperature, it was found that under conditions of 70-90 °C, the glucanase was completely inactivated within a very short period of time and did not participate in the production of reducing sugars [7]. It can be inferred that there are other pathways for reducing sugar production during black garlic processing.

After hydrolysis, the molecular weight of garlic neutral polysaccharides decreases significantly, the oligosaccharide content increases significantly, and the oligosaccharide content with a degree of polymerization below 10 increases from 15% to more than 75% [8]. Compared with the neutral polysaccharides in garlic, artificial gastric juice has a more significant effect on the hydrolysis of oligosaccharides. Oligosaccharides have low resistance, but they can significantly promote the proliferation of four species of lactic acid bacteria, lower the pH of the fermentation liquid, and have a stronger prebiotic effect. Fructooligosaccharides are a functional oligosaccharide that has a prebiotic effect and can promote the growth and function of beneficial intestinal microorganisms. However, the current process used to prepare black garlic has a limited fructooligosaccharide content, which greatly limits the prebiotic function of black garlic. Therefore, how to increase the fructooligosaccharide content by optimizing the processing technology is also one of the research directions.

1. 2 Organic acids

Organic acids in garlic play an important role in nutrient absorption, digestion and immunity. The acidity of garlic is 0.4% (in terms of lactic acid), and it does not taste sour. During heat treatment, the total acid content of black garlic continues to increase, giving it a sour taste. The Maillard reaction produces organic acids, mainly formic acid and acetic acid. The pH of the garlic changes from 6.42 (fresh garlic) to 5.00 (40 °C, 45 d) and 3.05 (85 °C, 45 d) [7]. The total acid content of black garlic varies greatly before and after processing, from 4.6 g/kg in fresh garlic to 33.61 (60 °C), 37.50 (70 °C), 30.96 (80 °C) and 36.37 g/kg (90 °C) in black garlic [6].

Citric acid, lactic acid, tartaric acid, oxalic acid and malic acid are the main organic acids in black garlic. Garlic extract contains organic acids such as citric acid, malic acid, lactic acid and fumaric acid, while black garlic extract loses fumaric acid and produces new acetic acid, formic acid, 3-hydroxypropionic acid and succinic acid (black garlic contains more formic acid and acetic acid) [9]. These changes are of great significance. An increase in the content of organic acids not only brings a sweet and sour taste, but also facilitates the hydrolysis of proteins and polysaccharides and the microbiological stability of black garlic. The reason for the increase in acidity after heat treatment of garlic is the consumption of a large number of basic groups (such as the amino group in amino acids) in the Maillard reaction and the formation of short-chain carboxylic acids.

1. 3 Sulfur-containing compounds

The main bioactive substances in garlic are sulfur-containing compounds. Allicin (S-allyl-L-cysteine sulfoxide), alliin (S-allyl-L-cysteine) and γ-glutamyl-S-allyl-L-cysteine are the main sulfur-containing compounds in garlic. In black garlic, the contents of allicin, alliin and γ-glutamylcysteine are 0.36%, 0.90% and 0.36% ~ 0.93% and 0.83% ~ 0.93% respectively.

deoxygallin, and γ-glutamylcysteine are 0.36%-0.90%, 0.36% ~ 0.93%, 0.83% ~ 2.83% [10]. Garlic has antibacterial, blood pressure lowering, blood fat lowering, anticancer, antitumor and other effects, which are related to the sulfur compounds in garlic. At the same time, sulfur compounds are also the main flavor substances in garlic, giving it a unique spicy flavor. During heating, the amino acids allicin and deoxyallicin in garlic degrade to form sulfur compounds such as allyl sulfide, some of which have a light aroma.

Black garlic mainly consists of 27 volatile sulfides, of which the higher content is 3-ethylidene-3,4-dihydro-1,2-dithiole (17.56%), diallyl disulfide (17.53%), 2-ethyl tetrahydrothiophene (13.24%), 2-vinyl-1,3-dithiane (8.81%), N,N'-dimethylthiourea and other compounds (8.00%). Compared to garlic, the contents of diallyl disulfide and diallyl trisulfide were significantly lower, which may be the main reason for the significant reduction in the pungent odor of garlic after heat treatment. After heat treatment, the content of 2-ethyl tetrahydrothiophene in black garlic was significantly higher than that in garlic, giving black garlic a light fragrance. The total volatile sulphur compounds in black garlic are slightly lower than in garlic, while pungent volatile substances such as diallyl disulphide and diallyl trisulphide are significantly reduced and aromatic compounds are increased [11].

The volatile characteristics of garlic and black garlic may vary due to differences in garlic variety, processing method and analytical method, but these results all indicate that after garlic is processed at high temperatures, the pungent odor is greatly reduced and the aroma is increased. Compared to garlic, the total amount of volatile sulfur compounds in black garlic also increases, which can effectively inhibit the synthesis of carcinogenic substances such as nitrosamines in the body, inhibit the formation and growth of cancer cells, lower blood pressure, resist aging, and prevent and treat diseases such as cardiovascular and cerebrovascular diseases.

1. 4 Polyphenols

Polyphenols are widely distributed in daily foods such as fruits, vegetables and cereals, and exhibit strong antioxidant properties. Garlic is one of the richest sources of phenolic compounds in foods. Tannins, flavonoids and phenolic acids are the main polyphenols in black garlic. During the processing, the polyphenols in black garlic are hydrolyzed by heating, producing a large number of small phenolic molecules and releasing more phenolic hydroxyl groups, which increases their relative content. Hydroxycinnamic acid derivatives are the phenolic acids with the highest content in garlic samples processed by different processes, among which the content of p-coumaric acid and o-coumaric acid has increased most significantly (by 14 times) [12]. After garlic is processed into black garlic, the total polyphenol content increases significantly by 7-11 times, the total flavonoid and total phenolic acid content increases by 1-5 times and 4-8 times respectively, making black garlic have stronger antioxidant activity and peroxide radical scavenging capacity than garlic [8].

1. 5 Amino acids

Amino acids are important nutrients in food. Their composition and content directly affect the nutritional value of food and are closely related to human taste. Garlic contains amino acids. After processing into black garlic, the content and type of free amino acids change significantly. As shown in Table 1 [13]. Fresh garlic is rich in free amino acids such as glutamine, asparagine and glutamic acid, as well as essential amino acids such as lysine, tryptophan and valine. After garlic is processed at high temperatures to make black garlic, the protein may denature, and some amino acids participate in the Maillard reaction, while others exist in a free state and constitute the nutrients in black garlic.

CHOI et al. [14] found that the content of all amino acids except leucine, isoleucine, methionine and phenylalanine decreased after processing. Cysteine is an important precursor of sulfur compounds in garlic and is also the parent of odor-producing compounds. The content of cysteine in black garlic is significantly reduced after heat processing, which may be related to the formation of the low-sulfur flavor of black garlic. The content of acidic amino acids such as tyrosine and aspartic acid, as well as basic amino acids such as glutamic acid, arginine and lysine, decreases with prolonged processing time. The content of polar amino acids such as threonine and serine and non-polar amino acids such as glycine and alanine also decreases. It is speculated that the decrease in these amino acids may be related to the Maillard reaction, which occurs between amines (usually amino acids) and carbonyl compounds (usually reducing sugars).

1. 6 Melanoidins

Melanoidins are brown macromolecular nitrogen-containing compounds that are formed during the late stages of the reaction by condensation and polymerization of Maillard reaction intermediates. In recent years, melanoidins have received increasing attention due to their antioxidant, prebiotic and anti-hypertensive effects.

Melanoidins were not detected in fresh garlic, while the total content of melanoidins in black garlic increased significantly when the fresh garlic was heated and processed into black garlic. The concentration was positively correlated with the number of heating days, and the color of the garlic increased at the same time. Processing garlic at 72, 75 or 78 °C for a period of time can lead to an increase in the color of the garlic (the Hunter L value of the entire garlic bulb decreased from 52.05 ± 0.38 to 18.01 ± 0.32, 18.04 ± 0.25 and  19. 06 ± 0. 26), while the intensity and rate of browning are highly dependent on temperature and processing time [15]. The absorbance was measured at 420 nm, and it was observed that the browning intensity of the samples processed at 85 °C increased rapidly until it reached a steady state on the 15th day, Garlic samples treated at 40–70 °C resulted in much slower changes in browning intensity (0.69 at 40 °C and 2.05 at 85 °C) [16].

The browning degree of black garlic is related to the progress of the Maillard reaction. The absorbance at 280, 320–360 and 420–450 nm corresponds to the products formed during the initial stage (glycosamin condensation and Amadori rearrangement), intermediate stage (sugar dehydration, fragmentation and Strecker degradation) and final stage (aldol condensation, polymerization and formation of heterocyclic nitro compounds). The products formed during these three stages follow a similar distribution, with a large number of intermediate products, as well as degraded compounds such as proteins, peptides and phenolic acids [17]. The increasing amount of melanoidins gradually causes the garlic sample to turn black, until it is a dark brown or black color.

Among the black pigments, the relative content of furans is the highest, followed by pyrroles, thiophenes, alkanes, furfural, phenols, etc. Black pigments have high chelating activity and antioxidant capacity, which is positively correlated with molecular weight. In vitro simulated digestion results show that black pigments are almost indigestible. Both α-amylase and hydrochloric acid treatment significantly reduced the metal ion chelating activity and antioxidant capacity of black garlic allicin, which could still be maintained above 60% after in vitro simulated digestion. This indicates that allicin has high bioavailability and bioaccessibility. Tests have shown that oral administration of melanoidins can significantly reduce weight gain and white adipose tissue mass in mice induced by a high-fat diet, and reverse glucose tolerance, especially at high doses. At the same time, after oral administration of melanoidins, the intestinal microbial environment of mice is improved, and bacterial diversity and abundance increase [18]. The above studies all indicate that black garlic black essence has great application potential. At the same time, black garlic black essence has great application potential as a dietary fiber in diabetes and obesity, and as an effective antioxidant, it can also be widely used in food additives or functional foods.

1. 7 5-HMF

5-HMF is a five-carbon cyclic aromatic aldehyde that can be formed by catalytic dehydration of reducing sugars (such as glucose or fructose) and amino acids during the high-temperature Maillard reaction, or by direct degradation of sucrose in an acidic environment. The formation of 5-HMF in food is highly dependent on processing and storage conditions, such as temperature and pH.

5-HMF, as a key intermediate product of the Maillard reaction, not only affects the biological activity of black garlic, but also its sensory effects. It can be used as an important indicator for predicting the processing rate of black garlic. Nuclear magnetic resonance hydrogen spectrum analysis showed that 5-HMF was not found in fresh garlic, while black garlic produces a large amount of 5-HMF during processing. The content of 5-HMF in black garlic extract obtained after heat treatment for 90 d increased by more than 6 times compared with that after heating for 25 d. LIANG et al. [9] used the amount of 5-HMF as a differential marker for fresh garlic and black garlic extracts obtained after heating for 5 and 25 d. The 5-HMF content in black garlic increases significantly during heat treatment, and the actual increase depends on the processing temperature. The higher the processing temperature, the faster the 5-HMF content increases. The 5-HMF level in black garlic prepared at 60 °C increases at a slow rate to 1.88 g/kg (about 0.39 to 0.46 times the 5-HMF in black garlic prepared at 70 °C, 80 °C and 90 °C). The 5-HMF content increases rapidly at 80 or 90 °C, but black garlic produces a bitter taste [19]. Freezing pretreatment can increase the 5-HMF content in black garlic by 25% (from 208.5 μg/g to 260.7 μg/g) [20].

However, there is still considerable debate about the safety of 5-HMF. High concentrations of 5-HMF are cytotoxic, irritating to human tissues and internal organs, and carcinogenic in the body. The formation pathway of 5-HMF during black garlic processing is still poorly understood, and effective methods for detecting 5-HMF levels during the processing of fresh garlic into black garlic require further research.

2 Biological functions of black garlic

After being heat-treated and processed into black garlic, garlic is easily absorbed by the human body. Black garlic is rich in functional ingredients such as polyphenols, sulfur-containing compounds, and melanoidins. Under the combined action of these compounds, black garlic has more powerful biological functions than fresh garlic.

2.1 Antioxidant and anti-aging effects

Antioxidant activity is the most prominent characteristic of black garlic. Black garlic has high DPPH radical, ABTS cationic radical, ·OH and ·O2- scavenging activities, thereby exerting an antioxidant effect. The enhancement of the antioxidant capacity of black garlic is closely related to the production of new antioxidant compounds. During the processing of black garlic, the increase or production of polyphenols (including flavonoids), β-carboline alkaloids, 5-HMF, and melanoidins, etc., all play an effective role in improving its antioxidant properties [21].

The allicin in black garlic can combine with lipids, and the combined product has the same function as vitamin E, that is, anti-aging and preventing atherosclerosis; the allinase in black garlic and its ethanol extract also have a certain anti-aging effect; the cysteine in black garlic can promote cell proliferation, and has a detoxifying and beautifying effect; black garlic is rich in germanium, which has a good anti-aging effect [13].

LEE et al. [21] fed 3-week-old diabetic mice with normal feed and freeze-dried ordinary garlic and black garlic, respectively, and measured the lipid peroxides and the activity of antioxidant enzymes in the liver after 7 weeks. The antioxidant capacity of black garlic was more than four times that of ordinary garlic. Compared with the control group of mice, the level of thiobarbituric acid reactive substances in the mice fed black garlic was significantly lower, and the activities of superoxide dismutase, glutathione peroxidase and catalase were significantly higher. CHOI et al. [14] found that the content of polyphenols and flavonoids in black garlic increased significantly within 21 days. The antioxidant capacity of black garlic was found to be significantly improved by the DPPH free radical and ABTS cation radical scavenging capacity. Both in vitro and in vivo experiments have proved that black garlic has strong antioxidant capacity and anti-aging effect.

2. 2 Antibacterial and anti-inflammatory effects

Allicin and allyl sulfide have a significant effect in the sterilization of black garlic and have a broad-spectrum antibacterial effect. In addition, the volatile components and leachates in black garlic have a significant inhibitory or killing effect on a variety of pathogenic bacteria. The minimum inhibitory concentration (Staphylococcus aureus and Escherichia coli) was significantly reduced, and a synergistic effect was observed [22]. KIM et al. [23] extracted 5-HMF from black garlic with chloroform and studied its effect on the expression of endothelial cell adhesion factors and monocyte adhesion in TNF-α-stimulated umbilical vein endothelial cells. The results showed that black garlic 5-HMF has anti-inflammatory effects and can be used as a potential therapeutic drug for vascular diseases such as atherosclerosis.

2. 3 Effect of regulating blood pressure, blood lipids, and blood sugar

With the improvement of living standards, the problem of “three highs” has gradually emerged and has become the “number one killer” of humans. Studies have found that black garlic has various effects such as lowering blood pressure, blood lipids, and blood sugar.

Ried et al. [24] used black garlic extract to assist in judging the blood pressure lowering effect of blood pressure lowering drugs, The results showed that black garlic had a better blood pressure-lowering effect. JUNG et al. [25] fermented black garlic using Saccharomyces cerevisiae and then fed three different doses of fermented black garlic to high-fat, obese mice. The same dose of Saccharomyces cerevisiae fermented black garlic was more effective than ordinary black garlic in combating obesity complications induced by a high-fat diet. High-fat rats fed different doses of black garlic extract were found to have lower SREBP-1C gene expression, which in turn down-regulates lipid and cholesterol metabolism, resulting in lower blood levels of total lipids, triglycerides and cholesterol [26].

Black garlic is effective in lowering blood sugar, has outstanding advantages, no side effects, and is also highly antioxidant. It can prevent complications of diabetes and is one of the first choices for hypoglycemic drugs. SI et al. [27] used Lactobacillus bulgaricus to prepare black garlic and conducted a clinical trial for gestational diabetes. After 40 weeks, fasting blood glucose and blood glucose levels 1 and 2 h after an oral glucose tolerance test were measured. The results showed that Lactobacillus bulgaricus promoted the conversion of pyranose to furanose glucoside, reduced fasting blood glucose and 1 and 2 h hemoglobin levels, and effectively improved gestational diabetes.

2. 4 Anti-cancer and anti-tumor effects

Studies have found that black garlic also has anti-tumor effects. Black garlic has shown excellent in vitro and in vivo therapeutic effects on cancers such as gastric cancer, liver cancer, lung cancer, leukemia, breast cancer and colon cancer.

In vitro tests have shown that black garlic extract causes dose-dependent apoptosis of GC-7901 human gastric cancer cells and can inhibit the in vivo growth of tumors in tumor-bearing mice. The increase in serum superoxide dismutase, glutathione peroxidase, IL-2 and spleen and thymus indices indicates that the anti-tumor effect of black garlic may be related to its antioxidant and immunomodulatory effects [28]. Black garlic aqueous extract has a significant growth inhibitory effect on liver tumors. After transplanting Kunming mice with H22 liver cancer cells, black garlic has an effective tumor inhibition rate of over 40% [29]. Black garlic hexane extract can significantly inhibit the proliferation of leukemia cells U937. The inhibitory effect is positively correlated with the concentration and the effect of apoptosis is positively correlated with the concentration of the active ingredient, and there is a certain dose-effect relationship. After treating U937 cells with 10 μg/mL black garlic hexane extract for 24 hours, the cell survival rate decreased by 60% [30].

Among the sulfur-containing compounds in black garlic, S-allyl cysteine (SAC) and S-allylmercapto-cysteine play a major role in its anticancer effect. In addition, the processing of black garlic converts the proteins in garlic into amino acids, which promotes absorption by the body and has a certain effect on improving the body's immunity, relieving fatigue, and preventing cancer and anticancer. Trace elements such as selenium and germanium in black garlic also have important anticancer effects.

2. 5 Protective effect on the liver and heart

Both in vitro and in vivo, black garlic has higher antioxidant and anti-inflammatory activity than garlic, which can provide a certain degree of protection to the liver and heart. Studies have found that black garlic has a protective effect on the liver in rats with chronic alcohol damage. Black garlic can improve lipid distribution and significantly reduce the levels of aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase and lactate dehydrogenase in the blood plasma [31]. In addition, black garlic is rich in SAC and polyphenols, which may exert a cardioprotective effect by relaxing the coronary artery during ischemia-reperfusion, thereby preventing ischemia-reperfusion-induced myocardial contractility decline [32].

2. 6 Immune enhancement

The body's immune function is achieved through the interaction of lymphocytes, monocytes and other related cells and their products. The fat-soluble volatile oil in black garlic can significantly improve the phagocytic function of macrophages in the body, effectively enhancing the immune system. At the same time, because the protein is converted to amino acids during processing, it can also effectively enhance the body's immune function. An increase in vitamin C content can also enhance the body's immunity.

Feng Yonghui et al. [33] gave mice intraperitoneal injections of black garlic extract solution for 5 consecutive days, and after killing the mice on the 6th day, they isolated and cultured their splenocytes and natural killer cell killing activity, the secretion level of NO in the spleen cell culture supernatant, and the levels of IL-2, IL-4, IFN-γ and TNF-α were measured, etc., to verify that black garlic extract can significantly increase the killing activity of natural killer cells and enhance the ability to monitor and remove abnormal cells in the body. In addition, black garlic extract can also increase the content of white blood cells, lymphocytes, and Lactobacillus rhamnosus, thereby enhancing the body's immunity [34].

2. 7 Other functions

Black garlic can also increase the total number of pyramidal neurons in the hippocampus of rats and improve spatial memory [35]; increase the number of Pucken cells in rats and improve motor coordination [36]; and adjust the balance of Th1/Th2 responses, inhibit the IG- 33-ST2 signaling pathway, and improve allergic asthma in mice [37].

3 Processing technology of black garlic

Currently, commercially available black garlic is mainly naturally formed through a series of chemical reactions and the Maillard reaction under high temperature and high humidity conditions. Microbial fermentation has the characteristics of being green and safe. With people's increasing health awareness, green bioconversion technology has gradually become a research hotspot. Some researchers have also used microbial fermentation in the processing of black garlic to give it better biological activity and physiological effects.

3.1 Non-fermented processing technology

At present, China's black garlic processing technology is mainly introduced from Japan and South Korea, and improvements are made to the original processing technology. At present, the black garlic processing technology is mainly a non-fermentation process, which is divided into solid-state processing and liquid processing.

3.1.1 Solid-state processing

Solid-state processing is currently the most common method of producing black garlic. Fresh, plump, whole garlic is selected and cultivated without the addition of any substances under high temperature and certain humidity conditions. The process flow of solid-state processing includes: garlic selection, peeling and removal of the first 1 to 2 layers of the stem, cleaning, grading and selection, high-temperature and high-humidity processing, sterilization and disinfection, packaging and other processes. Some studies have found that black garlic processed for 90 days is richer in nutrients than black garlic cultivated for 30 days. This may be because the short cultivation time means that there is less accumulation of active ingredients. Although the 30-day incubation period shortens the production cycle, the content of substances such as sugars and polyphenols is slightly lower than that of black garlic incubated for 90 days. However, the production cycle of black garlic incubated for 90 days is long, inefficient, and increases production costs. Therefore, how to adjust the process to obtain black garlic with high nutrient content while shortening the production cycle has become an urgent problem to be solved. Pretreatment and high temperature and humidity are two commonly used methods.

The effect of high-pressure pretreatment on the nutrient content of black garlic is significant, but the total sugar content and DPPH free radical scavenging capacity of black garlic after pressure-holding pretreatment are reduced, and the effect is not good [38]. Garlic was pretreated using two methods: low-temperature freezing and high-temperature boiling. The nutritional quality characteristics of the different pretreatment processes were compared. The results showed that compared with high-temperature boiling pretreatment and the control group of black garlic, the ripening time of black garlic pretreated by low-temperature freezing can be shortened by 4 d. In addition, the content of reducing sugar, total phenol and amino nitrogen in black garlic obtained by low-temperature freezing is higher, which is better than that of black garlic pretreated by high-temperature boiling and untreated black garlic [39].

Therefore, black garlic pretreated by low-temperature freezing has better quality. Zhu Xinpeng et al. [40] used microwave pretreatment to prepare black garlic and optimized the pretreatment process. Compared with black garlic without microwave pretreatment, black garlic prepared under optimal pretreatment conditions has significantly higher total phenol, reducing sugar and total acid content, no significant change in allicin, and a higher sensory evaluation. The use of methods such as respiratory inhibition, low-temperature freezing, and ultrasound to treat garlic can effectively increase the content of reducing sugars and amino nitrogen in black garlic, shorten the processing time of black garlic, reduce energy consumption, and improve functional ingredients.

Although the pretreatment methods for black garlic are not the same, they can all effectively shorten the processing time of black garlic, improve processing efficiency, and at the same time increase the functional ingredients in black garlic and enhance the functional effect. Therefore, pretreatment of garlic is necessary in the production and application of black garlic.

3. 1. 2 Liquid processing technology

Liquid processing refers to the crushing of fresh garlic into a paste and the addition of a certain proportion of water as a base for processing. The liquid processing process includes the following steps: garlic selection, peeling, cleaning, crushing, vacuum sealing, incubation in an incubator, drying, and packaging.

Cultivating black garlic under liquid culture conditions has a shorter processing time. Compared with garlic and black garlic cultivated in solid form, the total amino acid and individual amino acid content of black garlic cultivated in liquid form are increased, and the polyphenol content is also significantly increased. This shows that black garlic processed in liquid form has better antioxidant capacity, as also demonstrated by the DPPH radical scavenging rate test results. Luo Xue-cang et al. [41] found that the best process for liquid cultivation is a mass ratio of garlic paste to water of 2:1, a particle size of 4 mm, and a variable temperature of 70–80 °C. Under these conditions, the total phenol content of black garlic increases 5 times, and the superoxide dismutase activity increases 15 times. Black garlic is dark brown in color and has no garlic odor.

At present, there is relatively little research on the liquid fermentation of black garlic, but compared with traditional methods, the liquid processing method has the advantages of being simple, short processing time, low cost, high nutritional value and strong biological function.

3.2 Fermentation processing technology

In recent years, more and more researchers have shifted their focus to biotransformation. It is green and healthy, has no side effects of synthetic products, and also has a variety of biological activities and physiological effects. Microbial fermentation technology can increase the content of functional components in the fermentation products, such as polyphenols, proteins and peptides, dietary fiber, etc., and is a green and environmentally friendly means of bioconversion.

The biological activity of black garlic is enhanced by the fermentation process. JUNG et al. [25] used a two-stage fermentation method.

In the first stage, Saccharomyces cerevisiae was inoculated into a medium containing black garlic extract to increase the concentration of bioactive substances. After filtration, the culture solution was heated to extract and remove the cells. The solution was then evaporated and freeze-dried for storage. In mouse experiments, it was found that black garlic fermented with Saccharomyces cerevisiae had stronger in vitro antioxidant properties than black garlic prepared using traditional methods. It also showed stronger liver protection, kidney protection, blood lipid lowering and weight loss effects in diabetic and obese mice. In addition, it was found that garlic fermented with Bacillus subtilis contains a high level of stable nitrite [42]. Acute feeding of different doses of concentrated fermented garlic can effectively reduce the systolic blood pressure of spontaneously hypertensive rats, and the effect is dose-dependent.

Si et al. [27] used Bulgarian Lactobacillus to ferment black garlic. After fermentation, the garlic was rinsed with sterile water and dried in an incubator at 50 °C. The antioxidant properties of lactic acid bacteria-fermented black garlic were significantly improved, and it was effective in preventing gestational diabetes.

Bacillus species (e.g. Bacillus subtilis, Bacillus licheniformis) have strong acid-producing abilities. Adding a certain amount of Bacillus to the fermentation liquid can give foods unique flavours such as mellow, floral and fruity aromas [43]. Lactobacillus, the most common probiotic in the food fermentation industry, is already widely used in the food industry. Lactic acid bacteria can regulate the intestinal microecology in the human body, improve human cholesterol, regulate blood pressure, etc. After lactic acid bacteria fermentation, the ingredients in the raw materials will be biotransformed by lactic acid bacteria, which not only enhances the nutritional value of the raw materials (such as increasing the content of polyphenols, acids, dietary fiber, soluble protein and polypeptide content), improve the flavor and quality of the food (e.g., reduce the content of phytic acid), and also enhance the physiological activity of the functional ingredients in the raw materials (e.g., reduce blood sugar, blood pressure, and fight cancer) [44 - 45].

How to use the probiotic effect of microorganisms to enhance the biological activity of black garlic, improve the flavor and quality of black garlic, the selection of bacterial strains, the determination and optimization of the fermentation process, and safety issues all require further research.

4 Endophytic bacteria in black garlic

Endophytes are microorganisms that live in plant tissues for part or all of their life cycle. All healthy plant bodies in natural ecosystems coexist with endophytes, which secrete a variety of secondary metabolites to induce systemic resistance in plants. Therefore, plant endophytes can play a variety of roles such as biological control and yield-increasing agents.

Ji Yanru et al. [46] used conventional isolation and culture methods to study the law of change in the number of total bacteria and endophytic bacteria in black garlic during processing. There is no strictly meaningful fungi on the surface or inside the garlic, but there are a large number of aerobic and anaerobic bacteria. During the black garlic processing, the total bacterial and endophytic flora of the garlic first increased rapidly, then dropped sharply, and finally the number of microorganisms gradually tended to zero. When the temperature reached above 60 °C, the bacteria on the surface of the garlic basically lost their vitality, and only some endophytic bacteria survived. However, repeated temperature rises and falls gradually inhibited the activity of the endophytic bacteria until they lost their vitality. A melanin-producing bacterium was isolated at the 96th hour of black garlic processing and identified as Bacillus subtilis S8nyzx-1[47]. This bacterium can grow in garlic juice medium, grow vigorously, be heat-resistant, and produce melanin. It was inoculated into raw garlic cloves, and after 48 hours at 50 °C, the cloves turned black, while the uninoculated cloves were pale yellow, indicating that the bacterial solution had a certain effect on turning garlic from white to black.

Qiu et al. [48] used traditional culture methods to isolate and identify the endophytic bacteria of garlic and black garlic. The number of colonies from garlic was higher than that from black garlic, indicating that the microbial community has undergone some changes during the black garlic processing, with a slight decrease in the number, and that the endophytic bacteria (Bacillus subtilis, Bacillus methylotrophicus, Bacillus amyloliquefaciens) are the dominant bacteria in garlic and black garlic. This result is similar to that of Ji Yanru et al. [46].

However, the traditional method of culturing endophytic bacteria has some limitations, and some bacteria have limited culturability and reproducibility. Therefore, Illumina MiSeq sequencing technology (16S rRNA V3-V4 hypervariable region of bacteria) was used to increase the understanding of the endophytic bacteria in garlic and black garlic [49]. The results showed that the microbial community in black garlic was divided into 1,125 different genera in 45 phyla, dominated by four different genera: Thermus, Corynebacterium, Streptococcus and Brevundimonas. On day 0 of the black garlic fermentation, Proteobacteria, Firmicutes and Actinobacteria were the dominant phyla, accounting for 96.86% of the total. As the process progressed, the dominant phyla remained basically the same, but the relative abundances changed significantly. Specifically, the Proteobacteria decreased from 96.86% at 0 d to 44.53% at 12 d, but the Firmicutes, Actinobacteria and Bacteroidetes phyla increased from 1.04%, 0.67% and 0.18% to 20.47%, 8.84% and 8.54% respectively, indicating that the structure of the microbial community changed significantly after 12 days of heat treatment. During this process, the microbial diversity and abundance of the black garlic samples increased significantly. This result differs greatly from the results obtained by traditional culture methods, which proves that there are significant direct differences between the two methods.

Metabolic pathway prediction based on KEGG for 16S rRNA tagged gene sequences indicated that amino acid metabolism, carbohydrate metabolism and membrane transport play important roles during black garlic fermentation. Nutrients and bioactive substances in the garlic system are involved in the metabolic pathways of microbial communities at different stages, and interact directly or indirectly with substances produced by microorganisms. These effects are greatly influenced by processing conditions and ultimately determine the characteristics of the black garlic product. The newly formed functional compounds (such as oligosaccharides) play different roles in the growth of beneficial and harmful microorganisms. In short, there is extensive interaction between the key compounds in the garlic system and the coexisting microorganisms, and more research is needed, as these interactions further complicate the compositional changes in garlic during the conversion to black garlic.

The endophytic bacteria of garlic can ferment glucose, lactose, sucrose and garlic polysaccharides, are highly heat-resistant, and produce a large amount of organic acids and biologically active substances (such as extracellular polysaccharides). These active substances can significantly enhance the function of black garlic, extend its storage period, and improve its safety. indicating that the metabolic capacity of garlic endophytic bacteria has important biotechnological application value. However, the number of typical endophytic bacteria in garlic is relatively small, and it is necessary to isolate and expand them in vitro to explore their functions.

Therefore, using endophytic bacteria to accelerate the processing of black garlic, improve its flavor and functional substances, and extend its storage period will become a research hotspot in the future. Correlative analysis also showed that the diversity of the microbial community and some genera (such as Thermus and Bacillus) were significantly and positively correlated with the reducing sugar, total phenol and total acid content of black garlic. However, the role of microorganisms in the formation of black garlic quality still needs to be studied. In the future, the role of chemical reactions and microbial communities in the formation of black garlic quality during the black garlic cultivation process will be one of the research priorities through metabolomics and modern instrumental analysis and detection techniques.

5 Summary and outlook

After garlic is processed into black garlic, the main components such as sugars, organic acids, and polyphenols are significantly increased. The newly formed black essence and 5-HMF make black garlic have higher nutritional value and efficacy than garlic. Black garlic not only has good antioxidant and anti-aging effects, but also has protective effects on memory and the nervous system. It also has anti-cancer, anti-inflammatory, anti-allergic, hypoglycemic, liver-protecting and heart-protecting effects. Black garlic is gradually becoming popular as an emerging health food, and has a good market prospect.

At present, the main processing method of black garlic is natural cultivation under high temperature and high humidity conditions, mainly in solid form. Some researchers have realized that biotransformation not only does not have the side effects of synthetic products, but also increases the biological activity and physiological effects of the product. Using microbial fermentation technology makes the nutritional content of black garlic higher, improves the physiological functions of black garlic, and provides new ideas for the development of black garlic industrialization.

However, the following problems need to be further solved: (1) The selection of strains, the addition method and the addition process during fermentation are still immature, and the impact of different strains on the nutrients and physiological functions of black garlic and their mechanisms are still unclear; (2) The processing cycle of traditional black garlic is long, generally requiring 2 to 3 months or even longer under high temperature and high humidity conditions. How can the processing time of black garlic be shortened, production efficiency be improved, and energy conservation and emission reduction be achieved during the bioconversion process? (3) Researchers have used a combination of traditional culture and Illumina MiSeq sequencing technology to isolate and identify the endophytic bacteria in black garlic, and understand the changing trends of endophytic bacteria during the cultivation of black garlic. However, the impact of the presence of black garlic endophytic bacteria on the nutrients, flavor substances and biological activity of black garlic needs to be further explored; (4) As an emerging deep-processed garlic product, issues such as the formulation of production specifications, quality standards and safety indicators for black garlic deep processing also need to be urgently resolved.

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