Fractionation and Characterization of the Macromolecular Meaty Flavor Enhancer from Beef Meat Extract

Macromolecular meaty flavor enhancer was fractionated from a commercial beef extract. The macromolecular fraction was obtained by dialysis and separated by anion-exchange chromatography, Cu2+-chelate chromatography, and gel filtration chromatography. Two fractions were isolated as active meaty flavor enhancers. To elucidate the partial structures, the active fractions were treated with endoprotease Glu-C followed by high-performance liquid chromatography (HPLC) separation of the peptide fragments. Determinations of the amino acid compositions and amino acid sequences of the isolated fragments showed that the 2 active fractions consisted of collagen and tropomyosin. The macromolecular material obtained from heated collagen and tropomyosin in the low-molecular-weight fraction of beef soup stock enhanced the meaty flavor. These results suggested that collagen and tropomyosin were precursors of the macromolecular meaty flavor enhancer. Keyword: beef extract, meaty flavor, tropomyosin, collagen, heating process Introduction Beef extract is used in food manufacturing to generate a meaty aroma and meaty flavor designated as mouthfeel, continuity, and thickness. In a previous study (Shima and others 1998), the comparison of the taste between beef bouillon and a mixed reference solution was investigated. The mixed reference solution of beef bouillon was prepared from 36 chemicals (amino acids, nucleotides, sugars, organic acids, and others) on the basis of analytical data. It was reported that the reference solution lacked the brothy flavor designated as “brothy sour taste” and thickness. Furthermore, the authors purified the taste-active compound, which enhanced “brothy sour taste,” and identified the compound as N-(4-methyl5-oxo-1-imidazolin-2-yl)-sarcosine. This result suggested the existence of an unknown flavor enhancer in beef bouillon. Several studies have been carried out on the effects of macromolecular substances, such as gums and starches, on the taste of chicken broth (Rosett and others 1996). It was reported that the addition of these materials increased the intensity of thickness and decreased the intensity of saltiness of chicken broth. These results suggested that the addition of macromolecular materials could change the taste and flavor of foods. Although many studies have been performed on the taste of dior tri-peptides (Arai and others 1973; Hamilton and others 1974; Noguchi and others 1975), few have been conducted on the taste effects of longer peptides or proteinaceous materials. Recently, it was reported that a peptide fraction (MW 1000 to 10000) obtained from beef soup stock increased mildness and mouthfeel when it was added to beef soup (Ishii and others 1995). Furthermore, it was reported that several peptides from aged (2 °C for 1 wk) pork meat troponin-T decreased sourness when they were added to the soup prepared from non-aged pork meat (Nishimura 2001). However, there are few reports on the structure of the peptide or proteinaceous flavor enhancer. The present study examined the fractionation and the characterization of macromolecular substances from beef extract, which increased the intensities of the continuity, mouthfullness, and thickness designated as meaty flavor. Materials and Methods Beef extract and beef soup stock Commercial beef meat extract was purchased from Bordon Co. Ltd. (Sao Paulo, Brazil). The analytical data of the beef extract is as follows: moisture, 21.6% (w/w; dried at 105 °C for 4 h); total sugar, 6.6% (w/w); total nitrogen, 9.7% (w/w); NaCl, 2.8%; and creatinine, 7.5% (w/w). The beef extract was preserved for up to 1 y at 5 °C until used. Beef soup stock was prepared as previously reported (Kuroda and Harada 2000). Briefly, 6 kg of beef shank meat (obtained from market in Tokyo) was cut into pieces of 5 to 10 cm; 8 L distilled water was added and heated at 95 °C to 97 °C for 7 h. After removal of the beef meat, the soup was refrigerated for 16 h and the fat and the insolubles were removed by filtration. The soup stock obtained was freeze-dried and stored at –25 °C until use. Preparation of macromolecular fraction and lowmolecular-weight fraction One kilogram of beef extract or 500 g of beef soup stock powder was diluted with 4 L distilled water and dialyzed using a dialyzing tube (Sanko Junyaku, Japan) against distilled water. Both fractions were concentrated with an evaporator, freeze-dried, and kept at – 20 °C until use.