Thermal stability of kudzu root (Pueraria Radix) isoflavones as additives to beef patties
Kudzu root, Pueraria radix, extracts are a rich source of isoflavones. This study investigates the thermal stability of Pueraria radix extracts as a natural nutraceutical supplement in beef patties. The extract contained puerarin, diadzin, genistin, ononin, daidzein, glycitein, calycosin, genistein, formononetin and biochanin A; however, puerarin, daidzein and glycitein were the main components. The isoflavones concentrations in the spiked beef patties with kudzu root extracts were unaffected by cooking.
KeywordsKudzu root extract Beef patties Isoflavones Thermal stability
Phytoestrogens comprise a variety of structurally diverse chemicals, with flavonoids as their largest group. Isoflavones (dietary phytoestrogens) are one class of flavonoids that are available in various plant species mostly belonging to Leguminosae family. Isoflavones have a similar structure to β-estradiol in mammals. This structure allows the isoflavones to reduce or activate estrogenic activity in mammals by conferring with estrogen receptor on cells (Choi and Ji 2005; Delmonte and Rader 2006).
Kudzu (Pueraria lobata) native to Japan (called Kakkon) and China (called Ge Gen) has traditionally been used to relieve fever, promote salivation, relieve thirst, stop diarrhea, cure the common cold and relief wrist and shoulder stiffness (Chinese Pharmacopoeia Committee 2005; Sibao et al. 2007). Kudzu root (Pueraria radix) has been studied and found to contain high amounts of isoflavones, puerarin, diadzin, daidzein, genistin, genistein, formononetin and their derivatives (Delmonte and Rader 2006; Wu et al. 2011; Chen et al. 2001; Benlhabib et al. 2004). The kudzu root is widely used as a drug and highly valued for its medicinal properties (Fang et al. 2005). Studies have reported that kudzu root has anti-proliferative effects on cancer cells, antioxidant properties and hypoglycemic and antispasmodic effects. Reports also indicated that kudzu root improves blood circulation, prevents heart diseases, and reduces the urge to drink alcoholic beverages (Jun et al. 2003; Keung 2002; Choi and Ji 2005; Johnson and Loo 2000; Mazur et al. 1998; Patel et al. 2001). Kudzu root and its extracts are commercially available as dietary supplements in health food stores and from internet websites (Prasain et al. 2003; Delmonte and Rader 2006). The supplements are not regulated by Food Drug and Administration (Benlhabib et al. 2004). Commercial dietary isoflavones supplements often contain soy, red clover or kudzu extracts and in many cases combinations of these extracts from different sources (Delmonte and Rader 2006). Each plant has a different isoflavones profile. Kudzu root (Pueraria radix) has been shown to have a 10-fold isoflavones content compared to soybean (amounting to 2 g/ 100 g dry weight) (Choi and Ji 2005). Puerarin which accounts for about 80 % of isoflavones in kudzu is not found in soy-based products (Nakamura et al. 2000).
In the present market situation where consumers have an affinity for natural nutraceutical and a value added belief in natural nutraceutical, the food industry and researchers are trying to meet the demand of consumers. Food industry researchers are focused on the utilization of non-culinary/nutraceutical herbs or extracts with potential to function as healthy food ingredients. Researchers have used ginger, cinnamon bark, licorice root, rehmania root, peony root and angelica root to mask flavor in goat meat (Kim et al. 1993). Ethanol extracts of white peony, red peony, sappanwood, mountan peony, rosemary were used to reduce lipid oxidation in ground beef (Han and Rhee 2005). Kudzu root with its antioxidant, anti-aging property, benefits against cancer cells and other radical related diseases (Johnson and Loo 2000; Patel et al. 2001; Mazur et al. 1998) has the potential to be an important value-added ingredient in food products. The use of kudzu root extract in foods can prevent oxidation and increase shelf life. The sensory profile of kudzu root extract in a food model system is not available. Studies on sensory characteristics of soy isoflavones in food system disclosed the undesirable flavor characteristics of soy products. There is limited information available on kudzu root extracts (Lau et al. 2005; Rong et al. 1998) which contains isoflavones similar to those in soybeans. There are various opportunities to explore the kudzu root extract in a food model system. Studies of soy based isoflavones show that while isoflavones are stable when exposed to the heat, they can leach out into the cooking water (Uzzan and Labuza 2004). On the other hand there are researchers who have observed isoflavones to degrade at different temperatures in the food system and in the purified forms (Eisen et al. 2003; Uzzan and Labuza 2004; Xu et al. 2001). Baik et al. (2005) investigated the thermal stability and changes in the amount of isoflavones in kudzu root extract by heating at 80, 100, 121, 140, 165 and 180 °C for 90 min and found isoflavones diadzin and genistin to be stable till 121 °C and then they started to degrade thereafter. To our knowledge Kudzu root extract has not been studied in meat products such as beef patties. The aim of this study was to prepare a root tincture from the kudzu plant grown in the Southern states of North America, then to identify and quantify the isoflavones in the kudzu root crude extract, and to determine the thermal stability of isoflavones when added to beef patties.
Materials and methods
Acetonitrile, dimethylsulphoxide (DMSO), acetic acid, hydrochloric acid and ammonium hydroxide were obtained from J.T. Baker (Phillipsburg, NJ). Isoflavones were purchased from several suppliers. Daidzein, diadzin, genistein and genistin were purchased from LC laboratories (Woburn, MA). Glycitein, formononetin, puerarin were purchased from ICC Inofine (Hillsborough, NJ). Biochanin A and ononin were purchased from Sigma (St. Louis, MO). Calycosin was purchased from Chromadex (Santa Ana, CA).
Preparation of stock and calibration solutions
Each isoflavones stock solution was prepared by dissolving with DMSO in volumetric flasks. Five milligrams each of puerarin, diadzin, genistein, genistin, formononetin and biochanin A were dissolved to 5 ml with DMSO and 10 mg of daidzein, and glycitein were dissolved to 10 ml with DMSO. One milligram each of ononin and calycosin were dissolved to 5 ml with DMSO in separate volumetric flasks. The different amounts of isoflavones in each stock solution were due the limited availability of reference materials. Stock solutions were stored at -20 °C before being used.
The standard solution was prepared by mixing 1 ml of each 10 isoflavones in a volumetric flask and diluting to 20 ml with DMSO. The prepared standard solution was filtered through a 0.45 μm PTFE (polytetrafluoroethylene) membrane before HPLC analysis. The calibration curve for each isoflavones was obtained by adjusting the injecting volume to 5, 10, 20, 30, and 40 μl of standard solution accurately measured with gas tight volumetric syringes of HPLC (High Performance Liquid Chromatography). Plant sample preparation Kudzu root extract was prepared and supplied by Wild Pantry, Mothers Nature’s Super Store (Tellico Plain, TN). The kudzu root was dug in September, from the area in Monroe County, TN, between Tellico Plain, TN and Madisonville, TN. The fresh dug kudzu root was washed, cleaned and chopped into small pieces. Eight oz (226.80 gm) of chopped kudzu root were kept in a glass container with 16 oz (453.60 gm) of 40 % (80 proof) ethanol. A tight fitting lid was screwed tightly to prevent alcohol evaporation. The container was stored in a cool dry place out of direct sunlight, and turned upside down twice daily. After 2 weeks in storage, the extract was decanted into an amber bottle and shipped to the Department of Food Science, Mississippi State University (Mississippi State, MS). One milliliter of kudzu root crude extract was diluted to 5 ml, and 10 ml in volumetric flasks with DMSO. Five samples each of the 5 ml and 10 ml dilutions of the Kudzu root extracts were analyzed and compared to standard solutions prepared with reference materials. The crude kudzu root extracts were filtered through a 0.45 μm PTFE membrane before HPLC analysis.
HPLC was performed using Waters 2695 (Waters Association, Milford, MA) equipped with Waters 2996 photodiode array detector (PDA). A reverse phase column (Alltech Alltima HP C18 HL 5 μm, 250 × 4.6 mm, 1.0 ml/min) was used. The column temperature was kept constant at 40 °C. The UV wavelength was set at 203 nm. Samples were eluted at 1 ml/min with a linear gradient of 0.1 % MeCOOH in water (A) and 0.1 % MeCOOH in MeCN (B). The gradient was as follows: from 5 % B to 20 % B over 50 min, then from 20 % B to 40 % B over 40 min. At the end of run, the column was washed for 10 min with 70 % B before returning to its original condition. The complete run was 100 min, and the column was re-equilibrated for 20 min before the next run. The injection volume was fixed at 10 μl for all the analyses. Sample analysis was processed with Waters Empower Software 2002.
Peaks in the samples were identified by an automatic procedure based on the comparison of relative retention times (RRT) using the Empower software. The amount of isoflavones present in the samples was automatically obtained from HPLC, based on the calibration curve of individual isoflavones. The data obtained from HPLC was then back calculated to obtain the isoflavone content in the samples.
Preparation and cooking of beef patties with kudzu root extract
Fresh lean beef was obtained from Meat Science Laboratory, Department of Food Science and Technology, Mississippi State University. A random sample from each replication was analyzed to determine fat percentage (LabWave 9000™ Model FES. CEM Corporation. Matthews, NC). The fat and moisture content ranged between 7–8 % and 68–70 %, respectively in fresh lean beef. The fresh lean beef was ground using a 1.27 cm plate in a Hobart meat grinder (80055 Mixer-grinder, Hollymatic Co., Countryside, IL). Three 2 lb batches of coarsely ground beef were randomly assigned to 0 %, 1 %, and 3 % kudzu root extract batch. For each treatment group, ground beef was hand mixed with 1 % and 3 % kudzu root crude extract which was received from Wild Pantry and a non-treated control was made. The two treated batches and a control were stuffed (Risco I-36016 Thiene, Vincenza, Italy) into 7.62 cm diameter plastic tubes (Interstate Packaging, White Bluff, TN) and sealed at one end with metal clippers to make beef logs. The treated beef logs were labeled and frozen at −23 °C until further evaluation. All equipment was cleaned between each treatment for all replications. The exactly process was repeated to make samples for second and third replicates and the gap between the replicates was 1 week.
Cooking of patties
The frozen treated beef logs were removed from the plastic tubes and sliced into 1.27 cm thick patties (Patty maker, Butcher Boy ™ American Meat Equipment, LLC, Selmer, TN). Each patty was then placed in a vacuum bag (Prime Source, 15.24 cm × 21.59 cm, Kansas City, MO) and packaged using Model CV3HS, JVR Industries (Buffalo, NY) at 999 mbar vacuum for 1 s with a 2.5 s seal and returned to the freezer.
Later the frozen patties were thawed in the vacuum bags at room temperature for 15 min, weighed and then cooked by frying on a top stove griddle (Griddle 442A, Toastmaster Inc., Booneville, MO). Before frying the beef patties, the griddles were preheated to temperature of 176 °C for 5 min. Each patty was cooked for 5 min on both sides, with an additional 1 min on each side until an internal temperature of 71 °C was reached. This temperature was measured with a hand held digital thermometer. After cooling at room temperature, the cooked patties were kept at −23 °C before further processing for HPLC analysis.
Extraction of isoflavones from uncooked and cooked beef patties
The extraction of isoflavones from beef patties was performed as described by Vranova (2005), who quantified soy isoflavones in meat products by HPLC. The procedure was slightly modified to suit our objective. Heating (71 °C) of samples was not performed by Vranova. Two beef patties were randomly selected from either uncooked or cooked treatment and control groups (0, 1 % and 3 % kudzu root extract beef patties) and ground in a food processor (Cuisinart, Mini-Prep Plus, 3-Cup Food Processor, New York, NY) to obtain a homogenous mixture. Two grams of each homogenized sample was mixed with 20 ml of n-hexane in centrifuge tubes, then thoroughly vortex for 3 min and then sonicated at room temperature for 30 min in water bath (Branson 1200 E4, Branson Co, Danbury, CT). Next the samples were centrifuged for 10 min at 8,500 rpm, and the hexane was discarded. This procedure was repeated three times. The defatted meat sample was mixed with 10 ml of DMSO for dissolving isoflavones. The mixture was sonicated for 30 min and centrifuged at 8,500 rpm. The DMSO solution was pipetted out and filtered through 0.45 μm PTFE membranes before HPLC analysis for isoflavones. Two samples per treatment per replication were used for analysis.
Results and discussion
Identification and quantification of isoflavones in kudzu root extract
Isoflavone content (μg/ml dry weight) in kudzu root crude extract
Crude extract ± SD (n = 4)
3279.72 ± 261
23.8 ± 7.6
10.09 ± 6.4
10.97 ± 0.34
407.95 ± 204
51.96 ± 7.8
11.5 ± 0.25
31.94 ± 10.8
35.19 ± 12.49
7.245 ± 5.82
Stability of kudzu root extract isoflavones in beef patties during cooking
Effect of cooking on isoflavone content (μg/gm, dry weight) in uncooked and cooked beef patties formulated with kudzu root crude extract at 0 %, 1 % and 3 %
KRCE 0 %
KRCRE 1 %
KRCRE 3 %
The present study investigated the isoflavone in kudzu root extract prepared from the plant grown in southern states of North America. The research also focused on the stability of kudzu isoflavones in beef patties after cooking. The kudzu root extract contained ten isoflavones. The ten isoflavones were puerarin, diadzin, daidzein, glycitein, genistein, genistin, ononin, calycosin, biochaninA and formononetin. Puerarin and daidzein accounted for 95 % of the isoflavones content in the extract. The beef patties with kudzu root extract were analyzed to determine the thermal stability of isoflavones. There were four isoflavones, puerarin, daidzein, glycitein and genistein recovered from the beef patty samples. The other isoflavones were diluted to undetectable levels in the beef samples. The extractability percentage of isoflavones ranged from 65 % to 100 %. The isoflavone content in beef patties with 3 % kudzu root extract was significantly higher compared to samples with 1 % kudzu extract. No difference was observed in isoflavone content between uncooked and cooked samples. Cooking did not affect the quality or quantity of isoflavones in beef patties. This study introduces kudzu root extract as a functional ingredient in meat products while adding value and utilizing kudzu as a food source.
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