Method for Preparing Natural Nitrite
20240002887 ยท 2024-01-04
Assignee
Inventors
Cpc classification
International classification
Abstract
The present application relates to a method of producing natural nitrite, the method including preparing an extract from a plant containing nitrate, activating nitrate reductase within the plant extract, and inactivating the nitrate reductase. According to the method of producing the natural nitrite, nitrite may be produced in a high concentration from natural raw materials without inoculation of reducing bacteria.
Claims
1. A method of producing natural nitrite, comprising: preparing an extract from a plant containing nitrate; activating nitrate reductase within the plant extract; and inactivating the nitrate reductase.
2. The method of claim 1, wherein the plant is at least one selected from the group consisting of spinach, lettuce, Chinese cabbage, head lettuce, cabbage, young radish, sesame leaf, chicory, radish leaf, radish, crown daisy, kale, leaf mustard, leek, water parsley, and red beet.
3. The method of claim 2, wherein the plant comprises nitrate in a concentration of 3,000 ppm or more.
4. The method of claim 1, wherein the extract is obtained by squeezing or crushing the plant.
5. The method of claim 1, wherein the activating of the nitrate reductase comprises maintaining a pH range through a buffer system.
6. The method of claim 5, wherein the maintaining of the pH range comprises maintaining a pH within a range of pH 6 to pH 9.
7. The method of claim 1, wherein the activating of the nitrate reductase comprises fermenting the plant extract in a phosphate buffer solution or a carbonate buffer solution.
8. The method of claim 7, wherein the phosphate buffer solution is a buffer solution comprising NaH.sub.2PO.sub.4 and Na.sub.2HPO.sub.4, and the carbonate buffer solution is a buffer solution comprising Na.sub.2CO.sub.3 and NaHCO.sub.3.
9. The method of claim 1, wherein the inactivating of the nitrate reductase comprises heating the plant extract to a temperature of 55 C. to 95 C.
10. The method of claim 1, further comprising drying the plant extract after the inactivating of the nitrate reductase.
11. The method of claim 10, wherein the drying of the plant extract comprises lyophilization or spray drying.
12. A natural nitrite-containing composition prepared by the method of claim 1.
13. A composition for producing natural nitrite comprising a buffer solution containing a plant extract, wherein the plant extract contains nitrate.
14. The composition of claim 13, wherein the buffer solution maintains a pH of the composition within a range of pH 7 to pH 9.
15. The composition of claim 13, wherein the plant is at least one selected from the group consisting of spinach, lettuce, Chinese cabbage, head lettuce, cabbage, young radish, sesame leaf, chicory, radish leaf, radish, crown daisy, kale, leaf mustard, leek, water parsley, and red beet, and comprises nitrate (NO.sub.3.sup.) in a concentration of 3,000 ppm or more.
16. The composition of claim 13, wherein the buffer solution is a phosphate buffer solution comprising NaH.sub.2PO.sub.4 and Na.sub.2HPO.sub.4, or a carbonate buffer solution comprising Na.sub.2CO.sub.3 and NaHCO.sub.3.
17. A meat product comprising the natural nitrite-containing composition of claim 12 and meat, wherein the meat product comprises residual nitrite ion (NO.sub.2.sup.) of 70 ppm or less.
Description
DESCRIPTION OF DRAWING
[0056]
MODE FOR INVENTION
[0057] Hereinafter, the present application will be described in detail by Examples.
[0058] However, the following Examples specifically illustrate the present application, and the description of the present application is not limited by the following Examples.
Example 1
[0059] Production of Large Amount of Natural Nitrite without Using Reducing Bacteria
[0060] Unlike conventional methods of producing nitrite by using reducing bacteria that have the ability to reduce nitrate to nitrite, in the present application, a method of producing a large amount of natural nitrite by activating nitrate reductase contained in plants without using the reducing bacteria is newly invented.
[0061] [1-1] Confirmation of Effect of Nitrate Reductase in Increasing Nitrite Conversion Rate According to pH Control
[0062] Among plants containing a large amount of nitrate and having nitrate reductase, spinach was selected, and activation of its nitrate reductase was induced to produce nitrite. In addition to spinach, lettuce, Chinese cabbage, head lettuce, cabbage, young radish, sesame leaf, chicory, radish leaf, radish, crown daisy, kale, leaf mustard, leek, water parsley, and red beet may be used, and plants in which a concentration of nitrate is 3,000 ppm or more may be used.
[0063] With respect to the spinach, while pH conditions were changed, the activation of the nitrate reductase and fermentation was induced without inoculation of reducing bacteria from outside, and then the amount of nitrite was measured to identify whether the nitrate was converted to nitrite by the nitrate reductase within the spinach.
[0064] Specifically, Korean spinach (3,000 ppm or more of nitrate) was washed in running water to remove soil and foreign substances and then squeezed by using a conventional juicer for greens to thereby obtain a spinach extract. The spinach extract was fermented under a condition of impeller 200 rpm while pH conditions were changed, at a temperature of 20 C. to 40 C. in a 2 L fermenter (Marado-PDA, BIOCNS Co., South Korea), and a change in pH and changes in concentrations of nitrate and nitrite over time were measured. The amounts of nitrate and nitrite were analyzed by HPLC. A sample was diluted according to a detection concentration, and a supernatant thereof was filtered using a 0.25 m syringe filter so as to be used as an analysis sample, and Eclipse XDB-C18 (4.6250 mm, 5 m) was maintained at 40 C. and used as a column. A detector was a DAD detector and a 210 nm wavelength was used, a mobile phase A solution used was methanol, a mobile phase B solution used was 10 mM octylamine, and in a gradient mode, 0 min, 80% B, 15 min, 50% B, 20 min, 50% B, and 25 min, 80% B were flowed at a flow rate of 1.0 mL/min and injected in an amount of 20 L, for analysis. Standard products were manufactured with nitrate of 100 ppm or less and nitrite of 100 ppm or less, respectively.
[0065] pH Control Using Phosphate Buffer Solution System (NaH.sub.2PO.sub.4, Na.sub.2HPO.sub.4)
[0066] First, a phosphate buffer solution system including NaH.sub.2PO.sub.4 and Na.sub.2HPO.sub.4 was formed, the final pH of a buffer solution was adjusted to be 8.3, and buffer solutions with concentrations of 0.1 M and 0.2 M were each reacted with the fermented spinach prepared as described above. The initial pH of the squeezed spinach extract was 6.58, and the pH and nitrite conversion rate that were changed as a nitrate-to-nitrite conversion reaction progressed under the buffer solution system are shown in Table 1.
TABLE-US-00001 TABLE 1 Concen- 1 2 3 4 5 6 7 8 9 10 tration Item Extract hour hours hours hours hours hours hours hours hours hours 0.1M pH 6.58 7.42 7.42 7.33 7.26 7.10 6.90 6.65 6.48 6.35 6.13 Conversion 0% 1% 1% 2% 7% 17% 23% 41% 57% 57% 56% rate (%) 0.2M pH 6.58 7.55 7.47 7.47 7.35 7.23 7.05 6.82 6.61 6.50 6.41 Conversion 0% 1% 1% 1% 2% 6% 15% 23% 35% 42% 38% rate (%)
[0067] As a result, at the beginning of the reaction, the pH was measured to be about 7.5, but as the fermentation progressed, the pH gradually decreased, and a buffering effect of pH was shown in both 0.1 M and 0.2 M buffer solutions. In addition, in the case of the 0.1 M buffer solution, the nitrite conversion rate was measured to be 56% at 10 hours into the reaction. The 0.2 M buffer solution had a smaller change in pH and thus a better buffering effect than the 0.1 M buffer solution, but the nitrite conversion rate was measured to be 38% at 10 hours into the reaction, and thus, the reaction was delayed compared to the case where the 0.1 M buffer solution was used.
[0068] Given the above result, it was confirmed that the pH had a major effect on the activity of the nitrate reductase and the nitrite conversion rate, and accordingly, a buffer system capable of adjusting the appropriate pH should be applied in order to produce and obtain a larger amount of nitrite. In addition, it was confirmed that a large amount of nitrite could be effectively produced when the concentration of the phosphate buffer solution including NaH.sub.2PO.sub.4 and Na.sub.2HPO.sub.4 was adjusted and applied.
[0069] pH Control Using Carbonate Buffer Solution System (Na.sub.2CO.sub.3, NaHCO.sub.3)
[0070] Next, a carbonate buffer solution system (Na.sub.2CO.sub.3NaHCO.sub.3) was formed, the final pH was adjusted to be 9.5, and the spinach extract prepared as described above was fermented by using each of buffer solutions with concentrations of 0.01 M, 0.03 M, and 0.05 M. The initial pHs of the squeezed spinach extract were 6.21, 6.43, and 6.27, respectively, and the pHs and nitrite conversion rates that were changed as a nitrate-to-nitrite conversion reaction progressed under the buffer solution system are shown in Table 2.
TABLE-US-00002 TABLE 2 Concen- 1 2 3 4 5 6 7 8 9 10 tration Item Extract hour hours hours hours hours hours hours hours hours hours 0.01M pH 6.21 7.86 7.86 7.83 7.80 7.54 7.04 6.75 6.50 6.32 6.25 Conversion 0% 1% 1% 2% 2% 15% 26% 35% 43% 48% 52% rate (%) 0.03M pH 6.43 8.12 8.09 8.09 8.06 7.97 7.73 7.17 6.71 6.57 6.51 Conversion 0% 1% 1% 2% 2% 7% 36% 42% 75% 72% 72% rate (%) 0.05M pH 6.27 9.08 9.06 9.05 9.02 8.97 8.68 8.19 7.88 7.72 7.63 Conversion 0% 1% 1% 2% 2% 2% 2% 3% 6% 9% 13% rate (%)
[0071] As a result, in the case of the 0.01 M buffer solution, the pH decreased gently, and the nitrite conversion rate was measured to be 52% at 10 hours into the reaction. In the case of the M buffer solution, the pH was 6.51 and the conversion rate was measured to be 72% at 10 hours into the reaction, indicating the best nitrite conversion effect. In the case of the 0.05 M buffer solution, the pH was changed to strong alkalinity, deviating from the range in which the nitrate reductase is active and thus resulting in a significant decrease in the nitrite conversion rate.
[0072] Given the above result, it was confirmed that when the 0.03 M carbonate buffer solution was used, the nitrate-to-nitrite conversion efficiency was the best, and in spite of not inoculating reducing bacteria, a large amount of nitrite could be successfully produced. When a pH decreases due to metabolites that generate as a conversion reaction attributed to nitrate reductase progresses, the activity of the nitrate reductase may be inhibited, and the nitrite production efficiency may decrease. In the present application, a composition including a large amount of nitrite could be prepared by constructing a buffer solution system by which conversion to nitrite could be most efficiently achieved by solving these problems.
[0073] Optimization of Nitrite Conversion Reaction Conditions
[0074] Through the above experiments, it was confirmed that when the 0.03 M carbonate buffer solution system was used, the nitrite conversion rate was relatively higher, and thus, based on this, nitrite conversion reaction conditions were optimized, and a conversion rate according to a reaction time was measured and is shown in Table 3. The initial pH was within a range of 8.5 to 9.0, the M Na.sub.2CO.sub.3NaHCO.sub.3 buffer solution was used, and the reaction was performed at a stirring speed of 200 rpm.
TABLE-US-00003 TABLE 3 0 1 2 3 4 5 6 Extract hour hours hours hours hours hours hours pH 6.09 8.99 8.77 8.65 8.04 7.34 7.19 7.02 Nitrate 3,273 3,259 3,012 3,454 3,346 2,545 1,761 1,014 (ppm) Nitrite 0 0 0 0 297 933 1,556 2,082 (ppm) Conversion 0% 0% 0% 0% 9% 29% 48% 64% rate (%) 6.5 7 7.5 8 8.5 hours hours hours hours hours pH 6.83 6.74 6.70 6.60 6.54 Nitrate 438 51 0 0 0 (ppm) Nitrite 2,602 2,874 2,948 2,897 2,899 (ppm) Conversion 79% 88% 90% 89% 89% rate (%)
[0075] As a result, it was confirmed that the pH gradually decreased as the reaction progressed, but a buffering effect attributed to the buffer solution was sufficiently shown, and the conversion rate increased to about 90% at about 7 hours into the reaction, indicating a significantly excellent conversion effect.
[0076] [1-2] Confirmation of Nitrite Amount Increase Effect
[0077] In Example 1-1, the fermented spinach including a large amount of nitrite could be obtained by increasing the activity of the nitrate reductase within the spinach extract and the conversion rate by adjusting the pH conditions. The concentration of nitrite contained in the fermented spinach may be further increased through concentration and drying processes. In these processes, some nitrite may be lost, but it was confirmed which type of drying could obtain a high concentration of nitrite attributed to the least loss of nitrite by controlling drying methods and conditions.
[0078] First, an inactivation process was performed to remove the activity of the nitrate reductase in the fermented spinach prepared through the process of Example 1-1. After the fermented spinach was heated to a high temperature of 60 C. or more to inactivate the enzyme, 5% diatomaceous earth was added by using a filter press (JUNGDO 1000, JUNGDO Co., South Korea) and subjected to filtration using a 15 cc filter cloth for a first filtration, followed by a second filtration using a 5 m MF filter to additionally remove residual diatomaceous earth and fine substances. Next, sterilization was performed at a temperature of 95-100 C. for 1 minute and 30 seconds by using a UHT sterilizer. In addition, as concentration using a centrifugal thin-film concentrator was performed, a concentrate of a nitrite-containing composition of the present application was prepared (Table 4).
TABLE-US-00004 TABLE 4 Nitrite (ppm) Concentration concentration (27.5 Brix) 34,728
[0079] Next, powders of spinach extract were prepared through spray-drying and lyophilization methods by different spraying methods for the concentrate, and the concentrations of nitrite contained therein were measured and compared. In the case of spray drying, the solid content was adjusted to a level of 35-38 Brix by mixing maltodextrin with the concentrate. Ratios of the spinach concentrate to the maltodextrin were respectively set to 85:15, 88:12, and 90:10, followed by spray drying (in let temperature: 180 C., out let temperature: 90 C., sample injection speed: 10 mL/min). In the case of lyophilization, the concentrate was pre-frozen in a deep freezer at 70 C. without adding an excipient thereto and then subjected to using a lyophilizer. As conditions for the lyophilization, drying was performed for 72 hours with a vacuum degree of 20 kPa and with the temperature inside the chamber slowly raised from 40 C. to 30 C. The concentrations of nitrite included in the spray-dried powders (SD powders) in the above three ratios and the lyophilized powder (FD powder) were measured and are shown in Table 5.
TABLE-US-00005 TABLE 5 Composition Nitrite (ppm) SD powder (85:15) 67,806 SD powder (88:12) 72,659 SD powder (90:10) 80,780 FD powder (100) 104,917
[0080] As a result, in the case of the powders prepared through the spray drying, it was found that the lower the proportion of maltodextrin as the excipient, the larger the amount of nitrite. It was confirmed that when the amount of the excipient was small, the overall solid content decreased, and the yield also decreased. When the amount of an excipient is large, as the excipient is incorporated in a large amount, the total solid content may increase, and the fluidity of the spinach concentrate may decrease, resulting in differences in evaporation rates, leading to limitations on productivity. In contrast, the powder prepared through the lyophilization may have lower productivity and yield than the powder prepared through the spray drying, but since the lyophilization is a method of purely evaporating water without adding an excipient, a high amount of nitrite may be maintained, and as in the experimental result, it was confirmed that the powder prepared through the lyophilization had higher concentration of nitrite than other spray-dried powders.
[0081] Given the above result, it was confirmed that the lyophilization method could yield a large amount of powder having a higher concentration of nitrite than the spray-drying method, and it was confirmed that it was possible to prepare and use the powder in a form that could be used in a small amount such that other effects other than the function as a coloring agent could be minimized by using the high-concentration powder when the powder of the fermented spinach was to be applied to a product.
Example 2
[0082] Manufacture of Meat Products Using Nitrite-Containing Composition and Confirmation of Color Development
[0083] [2-1] Preparation of Model Meat Products
[0084] Using the fermented spinach powder prepared through Example 1, model meat products were manufactured by mixing pork hind leg meat with pork fat in proportions shown in Table 6. Preparation Examples 1 and 2 were prepared by adding the fermented spinach powder of Example 1 in concentrations of 20 ppm and 100 ppm, respectively, Comparative Preparation Example 1 was prepared without adding nitrite (negative control), and Comparative Preparation Examples 2 and 3 were prepared by adding synthetic nitrite in concentrations of 20 ppm and 100 ppm, respectively (positive controls). The fermented spinach powder was added to the meat products of Preparation Examples 1 and 2 in amounts of 0.03% and 0.149%, respectively, based on 100,000 ppm.
TABLE-US-00006 TABLE 6 Comparative Comparative Comparative Preparation Preparation Preparation Preparation Preparation Example 2 Example 3 Example 1 Example 2 Raw material Example 1 (20 ppm) (100 ppm) (20 ppm) (100 ppm) Pork 60.00 60.00 60.00 60.00 60.00 Pork fat 20.00 20.00 20.00 20.00 20.00 Purified water 16.70 16.70 16.70 16.60 16.50 Soy protein isolate 2.00 2.00 2.00 2.00 2.00 Refined salt 1.00 1.00 1.00 1.00 1.00 Phosphate 0.30 0.30 0.30 0.30 0.30 Synthetic nitrite 0.002 0.01 Fermented spinach powder 0.030 0.149 Total 100 100 100 100 100
[0085] [2-2] Measurement of Chromaticities of Nitrite-Added Meat Products
[0086] For the model meat products of Preparation Examples 1 and 2 and Comparative Preparation Examples 1 to 3, the appearances were observed and the chromaticities were measured. The meat products were pulverized with a blender and then put in a Petri dish, followed by performing measurement three times to measure Hunter L, a, and b values. For the surface color, a chromameter (CR-400, Minolta Co., Japan) calibrated with a standard white plate (L=97.40, a=0.49, b=1.96) was used. Based on the measured L, a, and b values, color difference (E) values were calculated and analyzed for comparison (Table 7). *E is the color difference from Comparative Preparation Example 1 (negative control), and **E is the color difference from Comparative Preparation Example (positive control) of the same nitrite concentration.
TABLE-US-00007 TABLE 7 Treatment group L a b *E **E Comparative 68.43 4.36 10.04 Preparation Example 1 Comparative 67.46 6.36 8.49 2.71 Preparation Example 2 (20 ppm) Preparation Example 1 67.85 7.69 8.26 3.81 1.40 (20 ppm) Comparative 68.34 7.84 8.25 3.91 Preparation Example 3 (100 ppm) Preparation Example 2 67.69 6.82 8.18 3.17 1.21 (100 ppm)
[0087] As a result of observing the appearances of the model meat products, the reddish color was barely observed in the negative control, Comparative Preparation Example 1, in which nitrite was not added, whereas the reddish color was observed in Preparation Examples 1 and 2 and Comparative Preparation Examples 2 and 3 even with naked eyes, compared to Comparative Preparation Example 1 (FIGURE). As a result of measuring the chromaticities, the difference in brightness between the meat products of Preparation Examples 1 and 2 and Comparative Preparation Examples was not large, and in the case of redness, except for Comparative Preparation Example 1 in which nitrite was not added, similar patterns were observed in Preparation Examples 1 and 2 and Comparative Preparation Examples 2 and 3. Even in the case of the color difference E value, there was a big difference from the negative control, Comparative Preparation Example 1, whereas there was no significant difference in color difference values between the positive controls and Preparation Examples 1 and 2.
[0088] Based on the above results, it was confirmed that although the fermented spinach powder prepared without using reducing bacteria was added to the meat products, the meat products had a color development effect at a level similar to that of meat products to which synthetic nitrite was added.
[0089] [2-3] Measurement of Residual Nitrite Ions
[0090] The amounts of residual nitrite ions in the model meat products of Preparation Examples 1 and 2 and Comparative Preparation Examples 1 to 3 were measured by using the diazotization method. 0.5 N sodium hydroxide, 12% zinc sulfate, pH 9.0 ammonium acetate, and distilled water were added to 10 g of each sample, followed by homogenization, heating, and filtration processes to prepare a test solution. A sulfanilamide solution, a N-(1-naphthyl)ethylenediamine solution, and distilled water were added to the prepared test solution and reacted for 20 minutes. A spectrophotometer was used to measure, at a wavelength of 540 nm, the absorbance which was then substituted into a calibration curve to calculate the amount (g) of nitrite ion included in 20 mL of the test solution. Based on this, the concentrations of nitrite ions were calculated and are shown in Table 8. It is expected that the fermented spinach powder may be used in a minimum amount such that a color development effect may be sufficiently exhibited, and at the same time, residual nitrite ion may be minimized.
TABLE-US-00008 TABLE 8 Comparative Comparative Comparative Preparation Preparation Preparation Preparation Preparation Example 2 Example 3 Example 1 Example 2 Example 1 (20 ppm) (100 ppm) (20 ppm) (100 ppm) Nitrite ion (ppm) 0 4.0 35.0 8.0 27.0
Example 3
[0091] Analysis of Components in Nitrite-Containing Composition According to the Present Application
[0092] It was confirmed whether any component other than nitrite was included in the fermented spinach prepared through Example 1. A sample (hereinafter, fermented spinach) prepared by squeezing spinach and performing fermentation through pH control in Example 1-1, a sample (hereinafter, fermented spinach concentrate) subjected to inactivation and concentration processes as in Example 1-2, and a powder sample (hereinafter, fermented spinach powder) prepared through a lyophilization process as in Example 1-2 were used, and a fermented plant (hereinafter, fermented reducing bacteria) prepared by a conventional technique by inoculating nitrite reducing bacteria was used as a control. The conventional fermented plant was prepared by mixing dried celery powder with purified water and then inoculating reducing bacteria (Staphylococcus camosus) to perform fermentation.
[0093] [3-1] Analysis of Amino Acids
[0094] The types and amounts of amino acids contained in the fermented spinach, fermented spinach concentrate, and fermented spinach powder of the present application and the fermented reducing bacteria prepared through the conventional technique of the related art were measured (Table 9).
[0095] Specifically, 9.9 mL of distilled water was added to 0.1 mL of each sample solution, thoroughly mixed, and then centrifuged (10,000 rpm, 10 minutes, 4 C.), and the supernatant was filtered through a 0.25 m syringe filter. Amino acids of the filtrate were analyzed using an amino acid analyzer (high-speed amino acid analyzer, L-8900, Hitachi Co., Japan) under the following analysis conditions. A column used was 2622SC-PH ion exchange column (4.660 mm, Hitachi, Co., Japan). The mobile phase was in a gradient mode, and pump 1 pumped sodium acetate buffers (MCI buffer PH1, PH4, RG) at a column temperature of 57 C. and a flow rate of mL/min, and pump 2 pumped ninhydrin solutions (R1, R2) at a flow rate of 0.35 mL/min. The injection volume was 10 L, and a detector used was a dual-channel detector with channel 1: UV-570 nm and channel 2: UV-440 nm.
TABLE-US-00009 TABLE 9 Fermented Fermented Fermented Fermented spinach spinach reducing (ppm) spinach concentrate powder bacteria Aspartic acid 27.03 68.85 402.99 31.70 Threonine 38.17 ND ND 47.54 Serine 39.52 Trace 393.96 75.88 Glutamic acid 179.20 893.71 3,893.06 145.56 Glycine 30.93 159.99 492.11 ND Cysteine ND ND ND ND Alanine 62.52 535.07 1,693.99 18.85 Valine 109.11 891.48 2,710.17 32.81 Methionine 28.99 Trace 374.73 ND Isoleucine 47.83 593.67 1,322.64 ND Leucine 100.64 1,243.50 2,487.55 ND Tyrosine 50.69 Trace 1,366.18 ND Phenylalanine 134.44 2,362.49 3,318.69 74.82 Lysine 59.82 370.57 1,077.16 ND Histidine ND ND ND ND Arginine 12.73 ND ND ND
[0096] As a result, it was found that a total of 14 amino acids were produced and included in the samples such as the fermented spinach prepared in Example 1 of the present application, and some amino acids among them were modified during the concentration and drying processes or converted to other types of compounds during the metabolic process, resulting in some differences in the fermented spinach concentrate and the fermented spinach powder. Threonine was present in the control, fermented reducing bacteria, prepared with the technique of the related art by inoculating reducing bacteria, whereas threonine was not present in the samples such as the fermented spinach prepared through Example 1. In addition, glycine, methionine, isoleucine, leucine, tyrosine, lysine, and arginine were not detected in the fermented reducing bacteria, whereas all of the amino acids were detected in the fermented spinach of the present application, and all amino acids except for arginine were also detected in the fermented spinach concentrate and the fermented spinach powder. It was confirmed that the samples such as the fermented spinach included a large amount of amino acids such as aspartic acid and glutamic acid in addition to glycine, wherein glycine, aspartic acid, and glutamic acid are known as amino acids for umami, and thus, the nitrite-containing composition of the present application including such amino acids, when applied to meat or the like, may have positive effects not only in terms of color development but also in terms of taste.
[0097] Given the above result, it was confirmed that there was a clear difference between the fermented product prepared by inoculating reducing bacteria and the composition of the present application prepared by activating reductase of the spinach without reducing bacteria in terms of the composition of the amino acids included in the final composition due to the different preparing methods.
[0098] [3-2] Analysis of Nucleic Acid-Based Compounds
[0099] The types and amounts of nucleic acid-based compounds contained in the fermented spinach, fermented spinach concentrate, and fermented spinach powder of the present application and the fermented reducing bacteria prepared through the technique of the related art were measured (Table 10).
[0100] Specifically, 200 L of each sample solution in the filtered liquid state was obtained through centrifugation and injected into high-speed liquid chromatography (HPLC) device (HPLC, Waters, Milford, MA, U.S.A./Pump:Waters 510, Injector: Waters 712 WISP) to analyze the nucleic acid-based compounds. A UV detector (254 nm, Waters, Milford, MA, U.S.A.) and -Bondapack column (3.9300 mm) were used, and the analysis temperature of the column was 30 C. A 1% triethylamine solution adjusted to pH 6.5 using phosphoric acid was used as a mobile phase solution, and the speed was 1 mL/min.
TABLE-US-00010 TABLE 10 IMP.2 Hypox- AMP.2 Na .Math. 7.5 (ppm) Adenine anthine Na Guanosine Xanthine Inosine Xanthosine H.sub.2O Fermented ND Trace ND Trace 35.54 0.47 0.53 7.67 spinach Fermented ND 377.0 ND 45.0 577.0 115.0 ND ND spinach concentrate Fermented ND 984.0 ND 85.0 1,387.0 176.0 ND ND spinach powder Fermented 2.26 ND 11.8 ND ND ND ND ND reducing bacteria
[0101] As a result, adenine and AMP.2Na were detected in the fermented reducing bacteria prepared by inoculating reducing bacteria, but not detected in the fermented spinach, the fermented spinach concentrate, or the fermented spinach powder. In addition, it was confirmed that nucleic acid-based compounds such as hypoxanthine, guanosine, xanthine, inosine, xanthosine, and IMP.2Na.Math.7.5H.sub.2O, which were not detected in the fermented reducing bacteria, were produced and present in the fermented spinach, the fermented spinach concentrate, or the fermented spinach powder. It was confirmed that some compounds were included only in the fermented spinach and were removed during the concentration and drying processes, but hypoxanthine, guanosine, xanthine, and inosine increased in concentration.
[0102] Given the above result, it was confirmed that there was a clear difference between the fermented product prepared by inoculating reducing bacteria and the composition of the present application prepared by activating reductase of the spinach without reducing bacteria in terms of the types or amounts of the nucleic acid-based compounds included in the final composition since metabolic processes are different depending on whether the reducing bacteria were inoculated.
[0103] [3-3] Analysis of Organic Acids
[0104] The types and amounts of organic acids contained in the fermented spinach, fermented spinach concentrate, and fermented spinach powder of the present application and the fermented reducing bacteria prepared through the technique of the related art were measured (Table 11).
[0105] Specifically, 9.9 mL of distilled water was added to 0.1 mL of each sample solution, thoroughly mixed, and then centrifuged (10,000 rpm, 10 minutes, 4 C.), and the supernatant was filtered through a 0.25 m syringe filter. Thermo Scientific Dionex ICS-3000 system was used to measure the types and amounts of organic acids contained in each sample solution, and the analysis was performed under the following conditions. A column used was aminex HPX-87H ion exclusion column (7.8300 mm; Bio-Rad, Hercules, CA, U.S.A.). The mobile phase was in a gradient mode, and a solution of 0.005N sulfuric acid and acetonitrile (95:5, v/v) was flowed at a flow rate of 0.6 mL/min at a temperature of 33 C. and injected in an amount of 25 L. A detector used was a Conductivity Detector.
TABLE-US-00011 TABLE 11 Citric Malic Succinic Lactic Acetic acid acid acid acid acid Fermented ND ND 125.7 ND 1,027.1 spinach Fermented Trace Trace 723.1 Trace 12,402.9 spinach concentrate Fermented 2,360.4 1,966.9 5,349.2 1,203.4 45,393.7 spinach powder Fermented 87.0 1,274.2 29.2 149.3 243.7 reducing bacteria
[0106] As a result, adenine and AMP.2Na were detected in the fermented reducing bacteria prepared by inoculating reducing bacteria, but not detected in the fermented spinach, the fermented spinach concentrate, or the fermented spinach powder. In addition, it was confirmed that nucleic acid-based compound such as hypoxanthine, guanosine, xanthine, inosine, xanthosine, and IMP.2Na.Math.7.5H.sub.2O, which were not detected in the fermented reducing bacteria, were produced and present in the fermented spinach, the fermented spinach concentrate, or the fermented spinach powder. It was confirmed that some compounds were included only in the fermented spinach and were removed during the concentration and drying processes, but hypoxanthine, guanosine, xanthine, and inosine increased in concentration.
[0107] Given the above result, it was confirmed that there was a clear difference between the fermented product prepared by inoculating reducing bacteria and the composition of the present application prepared by activating reductase of the spinach without reducing bacteria in terms of the types or amounts of the nucleic acid-based compounds included in the final composition since metabolic processes are different depending on whether the reducing bacteria were inoculated.
[0108] [3-4] Analysis of Free Sugars
[0109] The types and amounts of free sugars contained in the fermented spinach, fermented spinach concentrate, and fermented spinach powder of the present application and the fermented reducing bacteria prepared through the technique of the related art were measured (Table 12).
[0110] Specifically, 9.9 mL of distilled water was added to 0.1 mL of each sample solution, thoroughly mixed, and then centrifuged (10,000 rpm, 10 minutes, 4 C.), and the supernatant was filtered through a 0.25 m syringe filter. Dionex ICS-5000 system was used to measure the types and amounts of free sugars contained in each sample solution, and the following analysis was performed. Columns used were CarboPac PA1 guard column (Dionex, 504 mm) and CarboPac PA1 analytical column (Dionex, 2504 mm) maintained at 30 C. A mobile phase A solution used was 400 mM NaOH, and a mobile phase B solution used was pure water. In a gradient mode, 0 min, 95% B, 5 min, 0% B, 30 min, 10% B, 30.1 min, 95% B, and 40 min, 95% B were flowed at a flow rate of 1.0 mL/min and injected in an amount of 5 L. A detector used was ECD.
TABLE-US-00012 TABLE 12 Trehalose Fructose Glucose Sucrose Lactose Fermented 12.45 27.63 ND ND Trace spinach Fermented ND ND ND ND 230.5 spinach concentrate Fermented ND ND ND ND 793.6 spinach powder Fermented ND 1,370.9 1,868.9 2,756.4 ND reducing bacteria
[0111] As a result, trehalose and lactose, which were not detected in the fermented reducing bacteria, were present in the fermented spinach, and in particular, lactose was included even in the fermented spinach concentrate and the fermented spinach powder, which had been subjected to the filtration and drying processes. In addition, as a result of the fermentation using the reducing bacteria, glucose and sucrose were present in extremely large amounts, but were not detected at all in the fermented spinach.
[0112] The fermented product prepared by inoculating reducing bacteria contains a large amount of reducing sugars as described above, and thus, may cause browning and negatively affect the appearance when applied to products to be sterilized at high temperatures. In contrast, the nitrite-containing composition of the present application has an excellent color development effect attributed to nitrite and has an effect of minimizing browning because a small amount of reducing sugars is included, and thus, when applied to meat products, may have an excellent synergistic effect in terms of improvement of the appearance quality.
[0113] [3-5] Analysis of -Aminobutyric Acids (GABA)
[0114] The amounts of -aminobutyric acids (GABA) contained in the fermented spinach, fermented spinach concentrate, and fermented spinach powder of the present application and the fermented reducing bacteria prepared through the technique of the related art were measured (Table 13).
[0115] Specifically, Water's AccQ Fluor reagent kit was used for derivatization for analysis. A reaction reagent was prepared by dissolving 1 mL of acetonitrile (vial 2B) in 6-aminoquinolyl-n-hydroxysuccinimidyl carbamate (vial 2A). 70 L of a borate buffer solution (vial 1) was added to 10 L of a standard solution and extract, vortexed, and then left at room temperature for 1 minute. After 20 L of the reaction reagent was added and vortexed, the mixture was reacted in a water bath at 55 C. for 10 minutes. After completion of the reaction, the mixture was cooled to room temperature and analyzed using HPLC (Waters 2690 system). A column used was Mightysil RP-18 GP column (4.6250 mm, 5 m, Kanto Chemical, Tokyo, Japan), a mobile phase A was obtained by diluting 200 mL of AccQ-Tag Eluent A concentrate in 2 L of HPLC water, and a mobile phase B used was AccQ-Tag Eluent B. Solvent composition was analyzed by gradient elution with an initial A:B maintained at 90:10, followed by 70:30 (0.6 mL/min) by 30 minutes, 0:100 (0.8 mL/min) by 31 minutes, 0:100 (0.8 mL/min) by 38 minutes, 90:10 (0.6 mL/min) by 39 minutes, and 90:10 (0.6 mL/min) by 50 minutes. The sample injection amount was 10 L, and a detector used was a fluorescence detector (474, Waters) with Ex. 250 nm and Em. 395 nm for analysis.
TABLE-US-00013 TABLE 13 Fermented Fermented Fermented Fermented spinach spinach reducing spinach concentrate powder bacteria GABA (mg/L) 54.0 940.0 2,150.0 10.0
[0116] As a result, it was confirmed that the fermented spinach contained at least 5 times higher concentration of GABA than the fermented product prepared using reducing bacteria, and the fermented spinach concentrate and the fermented spinach powder contained even larger amounts of GABA. It seemed that GABA was produced because some glutamic acid was reduced during the metabolic process when the spinach was fermented, and the fermented reducing bacteria included GABA in a concentration of 10 mg/L, which is not high, which is interpreted as a result of nitrite being produced through a different metabolic process.
[0117] In the above, although representative embodiments of the present application have been exemplarily described, the scope of the present application is not limited to specific embodiments as described above, and those skilled in the art will be able to make appropriate changes within the scope described in the claims of the present application.