CHEMICAL PROPERTIES

II. CHEMICAL PROPERTIES

A. Structural Relationships

1. Glutamate-Related Substances The L forms of α-amino dicarboxylates that have four to seven carbons possess taste

properties similar to that of l-glutamate (I) ( Fig. 1 ). Likewise, compounds that have the threo form and the hydroxy group in the β position, such as DL-threo-β-hydroxyglutamate (II), impart a more intense taste than those compounds that have the erythro form and

the hydroxy group in the γ position. L-Homocystate (III), which has an SO 3 H group in the γ position of the L-glutamate molecule, also has the umami taste. Other amino acid salts that have similar sensory properties are ibotenate (IV), tricho- lomate (V), and L-theanine (VI); in contrast, α-methyl-L-glutamate (VII), in which the α-hydrogen atom is substituted for a methyl group, is tasteless, and pyrrolidone carboxylic

acid (VIII), which is formed by the loss of water from the NH 2 and γ-COOH groups of L-glutamate, has a sour taste (Kaneko, 1938).

Table 3 Distribution of Nucleotides in Vegetable Foods

Nucleotides content (mg/100 g)

Welsh onion

Head lettuce

Green peas

Cucumber

Japanese radish

Bamboo shoot

Mushroom, shiitake

0 103

175

Dried mushroom, shiitake

French mushroom

0 trace

Dried french mushroom

0 trace

190

Mushroom, enokidake

0 32 45

Mushroom, matsutake

0 95 112

Mushroom, syoro

0 9 16

Mushroom, hatsutake

0 85 58

Mushroom, benitengu dake

0 0 trace

Mushroom, naratake

0 0 trace

Source : Hashida (1963,1964).

2. 5 ′-Nucleotides The relationship between umami taste and the chemical structure of nucleotides ( Fig. 2 )

has been systematically studied. As shown in Fig. 2, purine ribonucleotides having a hy- droxy group on the 6-carbon of the purine ring and a phosphate ester on the 5 ′-carbon of the ribose moiety impart umami tastes, whereas purine ribonucleotides phosphorylated at C-2 ′ or C-3′ of the ribose moiety are tasteless. Thus, IMP, GMP, and XMP (disodium xanthylate) have umami tastes. Purine deoxyribonucleotides having a hydroxy group on the C-6 of the purine ring and a phosphate ester on C-5 ′ of the deoxyribose moiety also have umami tastes, but their taste intensities are weaker than those of ribonucleotides. The phosphate ester linkage on C-5 ′ of the ribose moiety is necessary for imparting umami taste. The C-5 ′ phosphate must have both primary and secondary dissociation of the hy- droxy group to exhibit umami taste; if the hydroxyl is esterified or amidified, the umami taste is lost. Synthetic derivatives of nucleotides, such as 2-methyl-IMP, 2-ethyl-IMP, 2- N -methyl-GMP, 2-methylthio-IMP, and 2- ethylthio-IMP, are known to impart a stronger umami taste in the presence of glutamate than do normal nucleotides (Kuninaka, 1960).

B. Stability

1. Glutamate Glutamate is not hygroscopic and does not change in appearance or quality during storage.

The characteristic taste of glutamate, umami, is a function of its stereochemical molecular

Figure 1 Chemical structure of glutamate-related substances.

Figure 3 Dehydration of glutamic acid to pyroglutamate.

structure. The D-isomer of glutamate does not possess a characteristic taste or enhance flavors (Yoshida, 1978).

Glutamate is not decomposed during normal food processing or in cooking. In acidic (pH 2.2–4.4) conditions with high temperatures, a portion of glutamate is dehydrated and converted into 5-pyrrolidone-2-carboxylate (Yoshida, 1978).

At very high temperature, glutamate racemizes to DL-glutamate in strong acid or alkaline conditions, but especially in the latter. Maillard (or browning) reactions occur when glutamate is treated at high temperatures with reducing sugars, as is the case with other amino acids (Fig. 3) (Yoshida, 1978).

2. 5 ′-Nucleotides IMP and GMP are not hygroscopic. IMP and GMP are stable in aqueous solution, but in

acidic solution at high temperature, decomposition of the nucleotides occurs. The ribose linkage of 5 ′-nucleotides is more labile than the phosphomonoester linkage, and the purine base is completely liberated by heating at 100 °C in 1 N HCl.

Enzymatic activity can also have a significant influence on flavor enhancer break- down and buildup. The phosphomonoester linkage of 5 ′-nucleotides is easily split by phos- phomonoesterases, which are readily found in plant and animal products. From a practical standpoint, these enzymes should be inactivated before the addition of 5 ′-nucleotide flavor enhancers to foods. Heating or storage below 0 °C is usually sufficient to cause inactivation.

C. Manufacturing Process

Glutamate and 5 ′-nucleotides were originally isolated from natural sources. Even today certain flavor enhancers can be economically isolated from various natural products, but certainly not in the quantities required by the food industry.

Presently the vast majority of commercial MSG is produced through a fermentation process: Most L-glutamic acid producing bacteria are gram-positive, non–spore forming, and nonmotile and require biotin for growth. Among these strains, bacteria belonging to the genera Corynebacterium and Brevibacterium are in widespread use along with an oleic acid requiring auxotrophic mutant, which was derived from biotin-requiring Brevibacter- ium thiogenitalis .

These bacteria can utilize various carbon sources, such as glucose, fructose, sucrose, maltose, ribose, or xylose, as the substrate for cell growth and L-glutamic acid biosynthe- sis. For industrial production, starch (tapioca, sago, etc.), cane molasses, beet molasses,

An ample supply of a suitable nitrogen source is essential for L-glutamic acid fer- mentation, since the molecule contains 9.5% nitrogen. Ammonium salts such as ammo- nium chloride or ammonium sulfate and urea are assimilable. The ammonium ion is detri- mental to both cell growth and product formation, and its concentration in the medium must be maintained at a low level. The pH of the culture medium is very apt to become acidic as ammonium ions are assimilated and L-glutamic acid is excreted. Gaseous ammo- nia has a great advantage over aqueous bases in maintaining the pH at 7.0–8.0, the opti- mum for L-glutamic acid accumulation. It serves as a pH-controlling agent and as a nitro- gen source, and solves various technological problems.

Moreover, recent technological innovations, such as genetic recombination, cell fu- sion, and bioreactor development, are now being applied for further improvement of L- glutamic acid fermentation. Genetic recombination and cell fusion techniques might be useful for the genetic construction of microorganisms with higher production yields or

with the capability to assimilate less expensive raw materials such as C 1 compounds and cellulosic materials. Bioreactors packed with L-glutamic acid producing microorganisms are being investigated in an attempt to improve productivity (Hirose et al., 1985).

IMP and GMP are commercially produced by two procedures: (1) degradation of RNA with 5 ′-phosphodiesterase to form 5′-nucleotides, and (2) fermentation, resulting in the production of nucleosides, which in turn can be phosphorylated into 5 ′-nucleotides.

MSG, IMP, and GMP occur as colorless or white crystals or as white crystalline powders. They are odorless and dissolve in water readily.

D. Assay Techniques/Analysis for Flavor Enhancers in Food

1. Glutamate As glutamic acid is the predominant amino acid in most proteins, excessive protein dena-

turation can result in significantly higher measured glutamic acid levels than can be attrib- uted to actual glutamate addition.

Paper and thin-layer chromatography, amino acid analyzer procedures, gas chro- matographic measurement of the trimethylsilyl ether derivative of glutamic acid, and po- tentiometric titration methods are available. Enzymatic analysis has also been conducted utilizing L-glutamate decarboxylase from pumpkin rind or Escherichia coli and L-gluta- mate dehydrogenase, which catalyzes the conversion of L-glutamate to α-ketoglutarate (Schanes and Schanes, 1946).

In general, glutamate is extracted from the food, preferably under acidic conditions, as free glutamic acid and is subjected to quantitative analysis using liquid column chroma- tography. A typical analytical method using amino acid analyzer is detailed below (Fujii et al., 1982).

a. Preparation of Sample Solution water-based or soluble food products. Weigh accurately an amount of sam- ple corresponding to 50–100 mg of glutamic acid and dissolve or disperse it in water. The total volume of the sample solution should be adjusted to 200 mL accurately.

Take about 2 mL of this solution and add to 10 mL of 1 g/dL picric acid solution. For a suspension, perform the following deproteinization operation. For a nonsuspension, use this solution as is.

Deproteinization operation . Take accurately 50 mL of the sample solution, add Deproteinization operation . Take accurately 50 mL of the sample solution, add

50 mL for sample solution. protein food products. Weigh accurately an amount of sample corresponding

to 50–100 mg of glutamic acid, place into centrifuge tubes, and add about 5 mL of water at about 100 °C for each 1 g of sample. Heat in a water bath for 15 min, cool, centrifuge under refrigeration for about 10 min at about 5000 rpm, and decant the supernatant solu- tion. Repeat the same procedure with 3.3 mL of hot water (100 °C).

Repeat this operation three times. Combine all of these separate supernatants quanti- tatively, add water to a final accurate volume of 200 mL, and filter as needed for sample solution. If the total of the combined supernatants exceeds 200 mL, concentrate under reduced pressure until a volume of approximately 180–190 mL is reached; then add water to an accurate volume of 200 mL, and filter the sample solution if necessary.

Take about 2 mL of this sample solution and add to 10 mL of 1 g/dL picric acid solution. In the case of a suspension, perform the deproteinization operation described under Water-Soluble Foods on the sample solution. If the sample solution is clear, this operation is not needed.

fatty foods. In general, perform the same operation as described under protein food products for the sample solution. However, if there is separation into oil and water layers or if there is marked suspension, perform the following defatting operation for sample solution.

Defatting operation . Place quantitatively all the separate solutions into a separating funnel and add 50 mL ethyl ether or ethyl ether–n-hexane (2 : 1) mixture for each solution. Shake well. Repeat the defatting operation two times and separate out the water layer. Heat the water layer in a water bath until the ethyl ether or n- hexane odor disappears, and add water to make an accurate total volume of 200 mL; filter, if necessary.

b. Preparation of Standard Solution. Weigh accurately 127.2 mg of monosodium L- glutamate monohydrate, and dissolve in water to an accurate volume of 100 mL. Take accurately 2 mL of this solution and add citric acid buffer solution (pH 2.2)* to an accurate total volume of 100 mL for the standard solution.

c. Method of Assay measurement conditions. Using the amino acid automatic analyzer for liquid

chromatography, measure under the following conditions: Packings: gel-form strong cation-exchange resin; average grain size 17 µm; cross-

linkage rate 8% Column dimensions and column temperature: 500 mm ⫻ 9 mm, 55°C

Eluent: citric acid buffer (pH 3.25),† 0.6 mL/min

* Preparation of citric acid buffer (pH 2.2): Dissolve 19.6 g of sodium citrate dihydrate, 16.6 mL of hydrochloric acid, 0.1 mL of n-caprylic acid, 20 mL of thiodiglycol and BRIJ-35 solution in water and adjust to 1000 mL. † Preparation of citric acid buffer (pH 3.25): Dissolve 7.7 g of sodium citrate dihydrate, 17.9 g of citric acid monohydrate, 7.1 g of sodium chloride, 0.1 mL of n-caprylic acid, 5 mL of thiodiglycol, 1 mL of BRIJ-35

Reaction coil: 20 m ⫻ 0.5 mm Reaction bath temperature: 98 °C

Ninhydrin solution‡ flow rate: 0.3 mL/min Injection volume: 500 µL Wavelength: 570 nm

preparation of test solution. Take accurately 10 mL of the sample solution, add hydrochloric acid solution (1 → 6), adjust the pH to 2.2, and make up accurately 100 mL with citric acid buffer (pH 2.2) for the test solution.

determination. Calculate the amount of L-glutamic acid by the following for- mula:

W S Amount of L-glutamic acid (%) ⫽ A

⫻ 0.03145 WA S

where W S ⫽ milligrams of standard monosodium L-glutamate monohydrate, W ⫽ grams of sample, A S ⫽ the L-glutamic acid peak area of the chromatogram obtained using the standard solution, and A ⫽ the L-glutamic acid peak area of the chromatogram obtained using the test solution.

Amount of monosodium L-glutamate monohydrate (%) ⫽ amount of L-glutamic acid (%) ⫻ 1.272

2. 5 ′-Nucleotides In the case of 5 ′-nucleotides, there are numerous procedures that utilize paper and thin

layer chromatography and high-performance liquid chromatography. Enzymatic assays specific for 5 ′-nucleotides are also available.

Thus it can be appreciated that a large number of techniques are available to measure flavor enhancer levels. Perhaps the most important limiting factor associated with all, or at least most, of these techniques is that they are not effective for a wide variety of foods, mainly because of incomplete extractions associated with certain foods or interference due to extraneous compounds. Thus, before a specific technique is chosen for a food, its potential limitations must be evaluated (Maga, 1983).