中国科技论文在线 Advanced Materials Research Vols

中国科技论文在线 http://www.paper.edu.cn Advanced Materials Research Vols. 197-198 (2011) pp 79-85 © (2011) Trans Tech Publications, Switzerland doi:10.4028/www.scientific.net/AMR.197-198.79

Some Characteristics to the Related Food Processing for Garlic Fructan

Xuesong Huang 1, a , Min Wang 1, b , Weibin Bai 1, c

1 Department of Food Science and Engineering, Jinan University, 601 Huangpu Ave W, Guangzhou 510632, China [email protected], [email protected], [email protected]

Keywords: Fructan,Garlic,Moisture Absorption, Foam Ability, Oil Absorption, Hygroscopy,Gel

Abstract. Garlic fructan (GF) is one of the main components of garlic ( Allium sativum L.) and has been largely produced from the residue discharged from getting essential oil in China. Some GF characteristics of the related food engineering, such as solubility, oil absorbability, foam ability, moisture absorption and retention, thermal and acidic stability and the like were investigated. Results showed that GF was soluble in water, slight soluble in ethanol but insoluble in acetone, ethyl acetate or butanol. The precipitation increased as the concentration of alcohol rise. GF presented better oil absorption than casein, as well as higher capacities of moisture absorption and retention in humid and dry environment than glycerin, but lower frothing and foam stability than egg white. GF was not stable at the environment of high acid and temperature. These results provide foundation for the application of GF in liquor, fat substitute food, chemical medicine and other use.

Introduction Garlic ( Allium sativum L.), belonging to the Liliaceae family, has attracted the much attention of plant physiologists and chemists [1-2] because of its unique flavor and medicinal functions. For example, the antioxidant properties [3], and therapeutic effects of garlic on atherosclerosis, hyperlipidemia, thrombosis, hypertension and diabetes [4] are well documented in Chian. Most of garlic’s prophylactic and therapeutic effects are ascribed to specific oil- and water-soluble organic sulfur compounds [5-6], which are responsible to the typical odor and flavor [7]. However, the garlic fructan (GF), which is the main component of garlic and made up 75 % of the dry matter [8], has been predicted to account for a significant portion of the pharmacological activities of crushed garlic. In the early 1940s the polysaccharides from garlic were investigated. In the following years the D-fructan was isolated and a linear inulin-type was postulated [9]. In 1981, the differences of the fructan content and synthesis in some Allium species were investigated. The degree of polymerisation (DP) was determined by means of size-exclusion chromatography [10]. Now, GF has been produced from the residue discharged from getting garlic essential oil in industrial scalar in China. It was found that garlic fructan belongs to grammine type and to the neokestose family with a molecular weight of 9000 ~ 10000 Da. DP postulated was about 58. However, Jack [11] extracted oligofructose and fructan from garlic by water and alcohol and analysed their molecular weight in the range of 1000 ~ 4500 Da with a composition of fructose: glucose = 15:1 . Numbers of reports showed the newly potential therapeutic effect of GF in human, such as its liver protection effect in injured mice serum or liver [12], inhibition of lens damages induced by UV irradiation [13], and therapeutic effect on Coxsackie B3 viral myocarditis [14] and so on.

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Meanwhile, GF as the carbohydrate source for lactic acid bacteria [15] makes it as potential prebiotics which may be used to produce the healthy food. However, it is necessary to reorganizate its adaptabilities profoundly for processing characteristics when GF is used in food engineering or pharmaceutical. There are few reports about the research on GF engineering characteristics so far. To set up foundation for GF application, this work described some common food characteristics such as solubility, capacities of oil absorption, moisture absorption and retention, stability in processing, frothing and foam stability so that it could produce high quality food or pharmaceutical excipient based on these engineering characteristics.

Materials and methods Raw Material and Reagents. Fresh garlic bulbs were purchased from local Shipai Market,

Guangzhou, China. Anhydrous ethanol, sodium hydroxide (N aOH), calcium phosphate

(C aHPO 4·2H 2O), ammonium sulfate [(NH 4)2SO 4], sodium carbonate (N a2 CO 3), hydrochloric acid, dinitrosalicylic acid, macro-resin, citric acid and its sodium salt were all purchased from Sigma Aldrich Inc. (St. Louis, MO, USA and Dongzheng Inc. Guangzhou, China) and all analytical purity. Casein, isolated soybean protein and peach gum were food grade and purchased from Dadi Food Additive Co. LTD (Guangzhou, China). Vegetable oil was afforded by Dongzhou oil Co. LTD. GF Preparation. GF was prepared by the method described in the patent [16]. In brief, garlic bulbs were subjected to mild pressure by hand to separate them into cloves. The cloves were crushed and homogenized in a comminuter with distill water. The solution was then gathered through filtration with four layers of gauze, followed by washing with distilled water and squeezing until no additional outflow of milky-white solution occurs. Garlic extract solution was then boiled at 100 ℃ for 20 min to remove the floating impurities before deodorized by chromatography (macro-resin as absorbance) and fined by using acidic silica sol and bentonite as described by Li [17]. The solution was cooled and centrifuged at 3500 r/min for 15 min to discard precipitation. The upper clear solution was collected and spray-dried and the GF powder was collected and used in the following experiments. Solubility of GF. Dissolve 1 g of GF in 10 ml organic solvents, such as ethanol, acetone, ethyl acetate or butanol, to observe the appearance of the mixture liquid. In order to know GF solubility in different ethanol, 2% GF solution prepared, then add 2 ml the solution into each test tube with 20 ml of ethanol in following concentrations (v/v): 10 %, 20 %, 25 %, 30 %, 35 %, and 40 % (v/v). Put them in fridge at 4 ℃ for a week. The precipitation was collected by filtration, dried at 60 ℃ and weighted. Solubility was calculated in 100 ml solution. Capacity of Oil Absorption of GF. Mix 2 g of GF with 10 ml vegetable oil for 30 sec in a Philip mixer (2 L,Royal Philips Electronics,Zhuhai,China). The mixed liquid was then allowed to stand at room temperature (28 ℃) for 30 min. They were thereafter centrifuged for 15 min 3,500 r/min, and the volume of the supernatant noted in a 10 ml graduated cylinder. The volume of dissociated oil in tubes was then observed, represented as V. Calculate the capacity of oil absorption (COA) as formulate below:

COA (ml / g) = (10 －V) /2………………………………………………… (1) Foam ability of GF. Accurately weiht 1.5 g of GF and 1.5 g of egg white and dissolve them in 50 ml distilled water separately, and their pH values were adjusted to 7.0 on Model 10 pH meter (Fisher Scient, Derver Instrument Company, USA). The initial volumes of the solutions were

recorded as V 0 before mixed. The GF or egg white solution was mixed well by the High Speed 中国科技论文在线 http://www.paper.edu.cn Advanced Materials Research Vols. 197-198 81

Organic Mixer (FHS-2A, Chongqing Jielun Science Technology Trade Development Co. LTD) for

1 min. The foam was transferred to a 100 ml measuring cylinder. The volume of foam (V α) at the following time after the stirring 10 min, 30min, 60 min, 90 min and 120 min was regarded as foam stability.

Foam ability (%) ＝(Vα- V0 / V 0 × 100 % …………………………………… (2) Hygroscopy of GF. Weight 0.5 g of GF (dried in drying cabinet till permanent mass) and put

them in the inner groove of Conway dish. Saturated (NH 4)2SO 4 and Na 2CO 3 solutions were added in the outer groove of two Conway dish to form an environment with relative humidity (RH) of 81 % and 43 %, respectively. The Conway dishes were then put in an oven which can maintain an invariable temperature at 25 ±0.2 ℃. The mass (M) of GF inside was weighed every 12 h to get the gained moisture weigh ( Δ M). As the blank, weigh 0.5g glycerin in another two Conway utensil and follow the same procedure as mentioned above. Hygroscopy (%) ＝ ΔM / M × 100 % …………………………………(3)

Moisture Retention Ability of GF. Weight 0.5 g dry samples and put them in the inner groove

of Conway dish then add 0.5 g (m 1) more distilled water and mixed them well. Saturated Na 2CO 3 solutions and dried silic gel were put in the outer groove of Conway dish to form an environment with relative humidity ( RH = 43%) and dried atmosphere, respectfully. The Conway dishes were then put in an oven which can maintain an invariable temperature as 25

±0.2 ℃. The mass of GF inside was weighed every 12 h to get the gained moisture weight (m 2). Glycerin in other Conway was selected as blank. Use the moisture survival rate (%) to show the moisture retention ability of GF:

Moisture survival rate (%) ＝ m2 / m 1 ×100 % ………………………… (4) Gel Formation Ability of GF. Prepare solution of concentration 20 %, 30 % and 40 % (W/V) in order to investigate whether the gel was formed or not. The water release was measured after freezing the solutions as described [18]. Consistence with Macromolecular Materials. Mix 2 ml of 2 % GF solution with 2 ml of 3 % macromolecular materials (Casein, soybean protein, CMC-Na and peach gum) solution, and then put the mixed liquid in KDC low sped centrifuge (Chuangxin Co., Ltd. Zonkia Branch,Hefei,China) to observe change in precipitation. Heat and acid treatment. Heat 2% GF solution at 90 ℃ and take out 2 ml of the solution every 30 min. The reducing saccharide parts were determined by dinitrosalicylic acid colorimetric method [19].The higher reducing saccharide, the more the decomposed GF. 2% GF solution was set at ambient temperature(20 ±5℃) and pH 2.01 adjusted with HCl by the pH-meter. Its reducing sugar was detected for every 30 min. Dada analysis. Triplicate samples for each treatment were taken. Each sample was analyzed individually in triplicate. The data were presented as mean and standard deviation of three determinations.

Results and analysis GF Solubility. Tests showed that GF is soluble in water, slight soluble in ethanol, but insoluble in acetone, ethyl acetate or butanol. Results showed different appearance when GF was mixed in diverse solvents: clear solution was formed in water; particles were formed in ethanol for the surface absorbed water quickly and cracked the inclusion; and precipitation was presented in acetone, ethyl acetate and butanol. 中国科技论文在线 http://www.paper.edu.cn 82 New and Advanced Materials

GF was slightly soluble in alcohol and the precipitation increased as the concentration of alcohol did. The appearance of the solution listed in Table 1 was similar but the solution in 10 % alcohol was clear completely for 2 weeks. However, there were 182mg/ml – 30mg/ml of GF dissolved in 10-40% alcohol solution. This result indicates that GF could be used as a nutrient substance in alcohol drink or healthy food, it might be better to add to low concentration alcohol for less precipitation. Table 1. Precipitation and solubility of GF in different concentration of alcohol Conc. of alcohol(V,V) 10 % 20 % 25 % 30 % 35 % 40 % Precipitation (mg) 0 15.4±0.660 18.5±0.901 23.8±1.12 28.2±1.11 33.4±1.32 Solubility(mg,100ml) 182 112±3.31 97.8±4.05 73.7±4.14 53.7±4.61 30.0±4.17

Capacity of Oil Absorption of GF. As shown in Table 2, GF’s COA is higher and is 1.26 time as casein. According to Eke’ point [20], high COA components improve flavor and increase the mouth feel of foods. Therefore, oil absorption is a significant factor in food formulations. Additionally, low-fat foods which contain high COA components as the substitute of high calore fat may reduce risk of obesity, coronary heart diseases and diabetes [18].Accordingly, high COA like GF is required in ground meat formulations, sausage, ham, yolk products, cheese, and other fat-containing foods. Besides, using this functionality, GF can be a potential substitute of the fat in the products to decrease the calories.

Table 2. Oil absorption capacity of GF and Casein *

GF Casein GF/ Casein Sample(g) 2 2 2 2 2 2 Oil(ml) 10 10 10 10 10 10 Dissociated Oil V (ml) 6.8 7.1 6.6 7.4 7.5 7.6 Oil Absorption Rate* 1.6 1.45 1.7 1.3 1.25 1.2 (ml/g) Average: 1.58±0.103 1.25±0.041 1.26 * Calculated by Equation (1) Frothing and foam stability of GF. GF is in certain frothing capacity with a foaminess rate of 50 % [calculated by Equation (2),see Table 3],which is less than egg white with the foaminess of 40 %.

Table 3. Compare of Foaminess (%) between GF and Egg White **

Time (min) 0 1 10 30 60 Foaminess of GF (%) 50±2 15±1 1±0.2 0.8±0.2 0 Foaminess of Egg white (%) 70±3 65±2 30±2 20±2 20±1.5 ** Calculate by Equation (2) According to Table 3, 20 % of foam stability for GF in 10 min after stirring, although there was still a certain amount of bubble left at the time of 30 min after stirring. Comparably, only 43 % of foam stability for egg white in 10 min, and 28 % of the foam still left even in 60 min. It indicates 中国科技论文在线 http://www.paper.edu.cn Advanced Materials Research Vols. 197-198 83

that GF owns some capacity in foaming and foam retention, though weaker than that of egg white. This characteristic may make GF to be used in the processing of ice-cream, butter, cream and the food in which it is incorporated into the foam mixture during the freezing process [21].

GF Hygroscopy The ambient relative humidity (RH) in our air environment is about 40 % ～80 % ，that was why the investigation was set in RH=43 % and 81 % to detect the hygroscopy and moisture retention ability of GF in this study. As shown in Figure 1 A, GF has a higher hygroscopy than glycerin in RH = 43 %. After 36 h, ability of moisture absorption was reducing for glycerin, but not for GF. Those results showed that GF’s hygroscopy was higher and could be a replacer of glycerin as a better moisturizer in daily cosmetic with its higher hygroscopy.

60

40 50

40 30

30

20 Hygroscopy % Hygroscopy 20 GF GF Hygroscopy % Hygroscopy Glycerin Glycerin

10 10 A 0 B 0 20 40 60 80 0 Hour 0 20 40 60 80 Hour

110 110

GF 100 Glycerin GF 100 Glycerin

90 90 Moisture Retention % Retention Moisture Moisture Retention % Retention Moisture 80 80

C C 70 70 0 20 40 60 80 0 20 40 60 80 Hour Hour Fig. 1 Hygroscopy and Moisture Retention Ability of GF compared to other substance A: Hygroscopy of GF and glycerin(RH=43%);B: Hygrosopy of GF and glycerin(RH=81%); C:Moisture Retention Ability of GF and glycerin;D:Moisture Retention Ability of GF and dried silica gel Nevertheless, Figure 1 B illustrated that GF had twice the hygroscopy higher than glycerin at the initial 12 h, but they came to a state of counterbalance after 72 h. In fact, speculated in Figure 1 A, hygroscopy was supposed to be higher for GF in the environment of RH = 81 %. Such an inconsistent result may result from the reasons that the glycerin used in this work was an analytical pure reagent without moisture added, but GF sample contained about 3 % of moisture, even if it was processed through drying [22]. That might reduce the ability of moisture absorption of GF. Moisture retention ability of GF. Figure 1 C and D showed that ability of moisture maintenance of GF was slightly lower than that of glycerin in RH = 43 %, but higher than glycerin in dry environment (around dried silica gel). This result indicated that not only absorb the moisture well, the GF can also maintain the water well even in dry environment. Generally speaking, GF has a good retention ability of moisture, which may be applied to prevent aging of bread and snack and to rotting of candies. 中国科技论文在线 http://www.paper.edu.cn 84 New and Advanced Materials

Gel formation ability of GF. Compared with inulin, which can form a kind of strong gel at a concentration of 30% (w/v), the tense GF solution did not form gel at even as high as 40 % (w/v) concentration. This study also found that when the GF solution was put in the fridge overnight, neither free water was released from ice crystal, nor precipitation was formed. This result suggested that GF’s molecular structure might be different from inulin [23] because it lack of ability of gel formation. Nevertheless, GF is stable in freezing environment without precipitation, which can be applied in ice-cream without change in structure. Consistence of GF with macromolecular substance. As shown in Table 4, solubility of macromolecular materials did not change too much after addition of GF. Moreover, less precipitation was left in soybean protein isolated solution. In general, GF can be mixed well with macromolecular substance and without any change in the solubility of the mixture, which provide basis information for the application of GF in some food like milk, soybean milk or gum, etc.

Table 4. Comparison of Before and After GF Addition in Macromolecular Materials

Casein SPI CMC- Na Peach Gum Before Clear solution 1.7 ±0.3 ml Precipitation Clear solution Clear solution After Clear solution 1.6 ±0.3 ml Precipitation Clear solution Clear solution GF Stability when treated at high temperature and acidity. GF could be decomposed when heated (see by Fig.2 A ).3.5% GF was decomposed at 90 ℃ for 30 min, and 14.5% decomposed for 120 min. Therefore, GF is not thermal stable and should be put as low temperature as possible in order to reduce thermolysis. As shown by Fig.2 B, GF was very prone to decomposed at pH 2.01 and ambient temperature (20 ±5℃).There are about 35% GF decomposed at pH 2.01 and ambient temperature (20 ±5℃) for 35 hours.

18

16 A 40 B Col 2 vs Col 3 - Col 5 14

12 30

10

8 20

6 Reduced sugar % sugar Reduced Reduced sugar % sugar Reduced

4 10

2

0 0 0 20 40 60 80 100 120 140 0 5 10 15 20 25 30 35 Heated time min Time h; pH=2.01 Fig. 2.GF stability A: Effect of heated time on hydrolysis; B: Effect of acid on hydrolysis

Conclusion Result presented above showed that the GF has good engineering characteristics. These mailly include that: ① GF is dissolved well in water and can be used in lots of food processing, slight soluble in ethanol and can be used in low alcohol drink; ②GF presented better oil absorption than casein, as well as higher capacities of moisture absorption and retention in humid and dry environment than glycerin;③ GF had lower frothing and foam stability than egg white; ④ GF was not stable at the environment of high acid and temperature;⑤ GF could be used in low alcohol and 中国科技论文在线 http://www.paper.edu.cn Advanced Materials Research Vols. 197-198 85

soft drink. These results provide processing or engineering foundation for the application of GF in liquor, fat substitute food, chemical medicine and the like.

Acknowledgements We are grateful to financially support for the Department of the Science and Technology of China (NO. 2007AA10Z340) and the Committee of the National Natural Science Foundation of China (NO. 30671472), to Dr. Xiyang Wu for critical comment and linguistic proof reading.

References [1] T. Baytop: J. of Istanbul Univ., Vol.6(1984),p.255. [2] F. U. Igene, S. O. Oboh, V. Aletor: J.of Food, Agric. & Environment, Vol.2(2005), p.28. [3] S. K. Banerjee, M. Maulik, S. C. Ansa: Life Sciences, Vol.12(2002) ,p.1509. [4] S. K. Banerjee, S. K. Maulik: Nutrition Res.,Vol.1(2000),p.1. [5] N. S. Kang, E. Y. Moon, C. G. Cho, S. Pyo: Nutrition Res. Vol.4(2001),p.617. [6] I. Krest: Journal of Nutrition, Vol.5(2001),p.171. [7] E. Block, S. Ahmad, J. L. Catalfamo: J. of Amer. Chem. Soc., Vol.22(1986),p.45. [8] W. Praznik, R. H. F. Beck, E. Nitsch: J. of Chromatography, Vol.2(1984),p.417. [9] N. D. Narendra, D. Amalendu: Carbohydrate Res., Vol.7(1978),p.155. [10] S. Baumgartner, G. Thomas, W. Dax: Carbohydrate Research,Vol.1( 2000),p.177. [11] N. L Jack, N. Shuryo: J. of Agric. & Food Chem., 11(1997),p.4342. [12] M. Zheng, S. Pan: J. of Xianning College(Medical Sciences),Vol.2(2003),p.85 (in Chinese). [13] M. Yang, M. Wang, Q. Hu: China Illuminating Engineering, Vol.6(1995),p4. [14] F. Cai, J. Wu, J. Chen: J. of Xianning College (Medical Sciences), Vol.2(2003),p.1 (in Chinese). [15] M. C. Paludan, R. Valyasevi, L.Huss: J. of Applied Microbiology,Vol.2(2002),p. 22. [16] X. Huang, W. Wang, A. Li, N. Zhang: China Patent, No.2003101176258(2003). [17] Y. Li, S. Shi, Y. Liu, X. Huang: Food Science and Techonology, Vol.7(2007),p.158 (in Chinese). [18] G. E. L. Nagar, G. Clowes, V. Kuri: Intern. J. of Dairy Technology,Vol.55(2002),p. 89. [19] Z. Zhang, X. Huang: Sci. & Tech. of Food Industry,Vol.8( 2009),p.321 (in Chinese). [20] O. S. Eke, E. N. T. Akobundu: Food Chemistry,Vol.4(1993),p.337. [21] N. Peron, A. Cagna, M. Valade, C. Bliard, B. V. Aguie: Langmuir, Vol.17(2001),p. 791. [22] G. P. Sharma, S. Prasad: J. of Food Engineering, Vol.50(2001),p. 99. [23] Y. Kim, M. N. Faqih, S. S.Wang: Carbohydrate Polymers, Vol.2(2001),p.135.