Quality Formation And Antioxidant Activity Of Black Garlic

China is an important garlic production country whose output accounts for 3/4 of the world’s total output. In some areas of China, Garlic industry has become a pivotal pillar industry to earn foreign currency and increase farmers’ income. However, our garlic products are mainly marketed domestically and internationally in forms of raw materials and the primarily processed products with low added value, volatile to acute inter-annual price fluctuation and severely confined to further development.However, black garlic is made of fresh garlic fermented under controlled high temperature and humidity, eliminating its smell and enhancing its antioxidant activity. At present, the quality of black garlic products is vary on the market, the processing time under high temperature is long and the energy consumption is high, which mainly due to the unclear mechanism of the garlic browning. Furthermore, the specific substance contributing to the strong antioxidant activity of black garlic is unknown. In this sense, it is scientifically vital to look into the characteristics and mechanism of quality formation and the functional substance for the antioxidant activity of black garlic, and to efficiently control the browning process, which can not only shorten processing time and reduce energy consumption but also promote the form of the black garlic functional materials and guarantee the quality, function and security of garlic products.In this study, we determined the growth and decline rule of the main quality substance in different stage of black garlic processed in different pre-treatments and different temperature. The antioxidant single material was obtained by silica gel column and semi-prep-HPLC. Analysis and structure identification was implemented by LC/MS, 1HNMR and 13 CNMR spectrometry. Then the antioxidant activity of the single material was tested by cell experiments.Black garlic was evaluated from the color, texture, taste, flavor and acceptability through sensory evaluation methods, the best quality indices of black garlic by 14-16 days processing were determined as the following: water(40%), reducing sugar(30%), total sugar(40%), total phenols(7-8mg / g), amino nitrogen(6%), 5-HMF(10 mg / g).Black garlic was produced in normal process after using freezing pretreatment and boiled inhibition pretreatment with Laiwu garlic as the processing material. The influence of pretreatment scheme was determined by monitoring the growth and decline rules of the main functional substances and the change rules of reducing sugar, total sugar, 5-HMF, total phenols, amino nitrogen and other substances. Reducing sugar, total phenols of black garlic increased during manufacturing process, which is 18.7%, 15.9% and 8.6%, 7.2% higher than the normal process, respectively. 5-HMF content of black garlic decreased after freezing and boiled pretreatment, which is 32% and 18.4% lower than the normal process. It shows that after the freezing and boiling pretreatment, it can generate antioxidant material in a larger extent, but reduce the content of 5-HMF in a maximum extent and bitter taste of the product, improving the taste and flavor of the final product. It shows that garlic cell was disrupted after freezing and boiling pretreatment, which benefits the saccharification of garlic and promotes the maturation of black garlic. It can generate antioxidant material in a larger extent, but reduce the content of 5-HMF in a maximum extent and bitter taste of the product, improving the taste and flavor of the final product.Black garlic was produced using air source heat pump system, we measured major quality indices including moisture, hardness, color value, 5-HMF, reducing sugars, total phenols and amino nitrogen at 60 oC, 70 oC and 80 oC. We also analyzed the change rule and its relationship with the quality formation of black garlic. Curing time of black garlic at 60 oC, 70 oC and 80 oC was 40 days, 25 days and 15 days, respectively. It was found that the hardness of garlic decreased more significantly at higher temperature. 5-HMF began to slowly accumulate from the tenth day at 60 oC to 13.25 mg/g after 40 days; but 5-HMF began to quickly accumulate from the fifth day to 13.45 mg / g after 25 days at 70 oC; 5-HMF began to accumulate shortly and reached 14.53 mg/g after 15 days at 80 oC. Total phenol of black garlic firstly increased and then decreased at 60 oC, 70 oC and 80 oC, showing the same trend, and ultimately reached 13 mg/g along with the extension of the aging time. But it accumulated the fastest at 80 oC, and then 70 oC and 60 oC. The amount of reducing sugar and amino nitrogen of garlic continue to increase at 60 oC, 70 oC and 80 oC, the higher the temperature, the faster the accumulation. They all ultimately reach about 42 g/100 g and 6.2 g/100 g with the extension of the aging time. High temperatures can accelerate the formation of black color and hardness, facilitating the accumulation of reducing sugar and the amount of amino nitrogen in the early processing stage; while low temperature can reduce the amount of 5-HMF, but increase the amount of total phenol and amino nitrogen, in favor of the quality formation of black garlic.The optimal extraction condition of total phenols in black garlic was confirmed by single factor and orthogonal experiments, and then antioxidant activity of black garlic was verified by in vitro antioxidant experiment. The best condition is the following: 80% ethanol is used as the leaching mention solvent, the ratio of material 80% ethanol is 1:5, the temperature of extraction is 80℃, the extraction time is 40 min. This experiment is aimed to further measure the oxidation resistance of black garlic according to the elimination of ABTS free radical, DPPH free radical, superoxide anion free radical and hydroxyl free radical. Compared with the normal garlic under the same condition and time, the antioxidant ability of black garlic is 10 times higher than the normal garlic. The strongest part of antioxidant ability was confirmed by antioxidant experiment of composition of extraction of n-butyl alcohol, extraction of ethyl acetate and extraction of water of black garlic. The result is the following: extraction using water >extraction using n-butyl alcohol >extraction using ethyl acetate.The compounds extracted from black garlic using ethyl acetate were separated by silica gel column and semi-prepare-HPLC, and seven constituents were isolated and purified from the crude extracts. Their structures were elucidated by means of physicochemical properties and spectroscopic, which are 5-HMF,(1R, 3S)-1-Methyl-1, 2, 3, 4-tetrahydro- β-carboline-3-carboxylic acid,(1S, 3S)-1-Methyl-1, 2, 3, 4-tetrahydro- β-carboline-3-carboxylic acid, lactic acid, uridine, adenosine and 2-acetylpyrrole.It is analyzed that the organic acids of black garlic are citric acid, lactic acid, tartaric acid, oxalic acid and malic acid by means of HPLC and the standard control.CAA experiment and anti-virus MTT study were performed with 5-HMF, carboline alkaloids, uridine, adenosine and freeze-dried samples of ethyl acetate extract, respectively. And the CAA value is about 65% of adenosine, about 69% of uridine, about 64% of 5-HMF, about 60% of carboline alkaloids, about 60% of ethyl acetate crude extract. From the above data we can conclude that ethyl acetate extract has a high antioxidant activity, which fit the results of in vitro experiments. The antioxidant activity of monomer also fit the antioxidant activity of the crude extract. The inhibition rate of adenosine is 42% on ES-2 and the inhibition rate of uridine is 47.9% on PC-3, but the inhibition rate of other substances on tumor cells is low. After analyzing the experimental data, the antitumor activity of ethyl acetate extract is not very significance.