Notes
Storage temperature and quality changes of natto
2021 年 27 巻 3 号 p. 497-504
詳細
2021 年 27 巻 3 号 p. 497-504
Storage conditions significantly affect the quality of natto, a fermented soybean food produced with Bacillus subtilis (natto). We investigated the relationship between the quality of natto and the storage temperature. Manufactured natto samples were stored at 5–20 °C for 20 days, and on every 5th day we evaluated the following: 1) viable bacterial number, 2) ammonia nitrogen, 3) free amino acids, 4) gamma-polyglutamic acid (γ-PGA), 5) hardness, and 6) color of soybean grains. During storage, natto products exhibited damage, especially when stored at 20 °C. Natto products were scored based on these quality indicators, and natto stored at 5 °C for 10 days or at 10 °C for 5 days had similar quality scores. Overseas export of natto is gradually increasing. The quality score proposed in this study is a basic indicator that can be used to rule out inferior products, and this will help to grow the natto market worldwide and facilitate distribution through a cold chain.
Overseas export of natto is gradually increasing i). Natto products are distributed domestically through cold chains. However, the global distribution network for natto is still poor and there are concerns regarding damage to natto due to unfavorable storage conditions during transport. In addition, the available information regarding quality changes of natto is limited. Therefore, systematic study of storage conditions is critical for quality control of natto.
Natto is a traditional Japanese soybean food made by fermentation with Bacillus subtilis (natto). After completion of fermentation, B. subtilis cells are still alive, and some extracellular enzymes remain active, which leads to damage associated with secondary fermentation. Natto products can exhibit unfavorable changes depending on storage conditions, such as color browning, weakened texture and stickiness, and development of ammonia odors. These quality issues are attributed mainly to extracellular proteases, including alkaline protease (AprE; also referred as subtilis NAT or nattokinase) produced by B. subtilis and Maillard reactions (Kada et al., 2013; Teodorowicz et al., 2018). However, the relationship between storage temperature and natto quality has not been examined. Further, long-term storage of natto under uncontrolled conditions leads to over-digestion of soybean proteins, which subsequently causes precipitation of tyrosine as white particles and accumulation of struvite (magnesium ammonium phosphate) as small diaphanous, glass-like fragments in natto products (Muramatsu et al., 1997). Currently, natto is usually kept at or below 10 °C for distribution domestically. Consequently, the tyrosine particles and struvite are seldom observed in Japanese natto.
In this study, natto samples manufactured by conventional methods were stored at 5–20 °C for 20 days, and on every 5th day, we evaluated the following six parameters for natto quality: viable bacterial number, ammonia nitrogen content, free amino acids, gamma-polyglutamic acid (γ-PGA), hardness, and color changes of soybean grains. We attempted scoring these values and proposed a practical benchmark for natto quality that may facilitate determination of freshness date, i.e., a best-before date to provide tasty natto for consumers.
Expansion of the freshness date for natto is feasible through cultivation of temperature-sensitive starter strains (Kitahara et al., 1989; Ishikawa et al., 2014), development of gas barrier packaging (Tamura and Murasawa, 1995a; Tamura et al., 1995b), genetic engineering of B. subtilis cells (Kada et al., 2008; Makino, 2007; Nishikawa, 2009), and pre-heating of natto products (Ogasawara and Ichise, 2014; Ichise et al., 2015). In addition, natto starter strains that produce less ammonia can be used (Kada et al., 2008; Takemura, 2014). The quality scoring proposed here can be used to quantitatively compare these approaches to expanding the freshness date for natto.
Manufacturing of natto samples In the present study, natto was made from soybean cultivar Yukishizuka as previously described (Kubo et al., 2011). Soybean grains were soaked in water at 20 °C for 15 h and steamed at 0.28 MPa (absolute pressure) at 131 °C for 25 min. An aqueous spore solution of seed strain (Miyagino) was inoculated in the steamed soybeans at 104 CFU/g, and 50 g of the inoculated sample was placed in each polystyrene package and covered by a polypropylene film with small holes. Fermentation was performed in a thermo-hygrostat (LH43-15p, Nagano Science Co. Ltd.) in a three-step program. In the first step, inoculated soybeans were kept at 39 °C under 98% relative humidity (RH) for 18 h and then held at 20 °C and 50% RH for 2 h in the second step. In the final step, inoculated soybeans were kept at 5 °C, 10 °C, 15 °C, or 20 °C for 5–20 days without humidity control prior to sampling.
Viable B. subtilis cell count Natto samples (20 g) and 180 mL of sterile saline (0.3 mmol/L of phosphate buffer [pH 7.2] containing 0.85% sodium chloride) were poured into each stomacher bag (Stoma Filter Type S, Central Scientific Commerce, Inc.), and the samples were crushed thoroughly for 1 min. The crushed samples were serially diluted with sterile saline, mixed with LB agar media kept at 50 °C, and then poured into petri dishes. Plates were incubated at 35 °C for 48 h for colony counting.
Ammonia nitrogen content Natto samples (1 g) were strained twice through a strainer (30 mesh). Then, samples were measured in a measuring flask which was filled with distilled water up to 50 mL and mixed by vortexing to dissolve the natto. The ammonia nitrogen content of natto was determined using the LabAssay Ammonia kit (FUJIFILM Wako Pure Chemical Corp.) according to manufacturer instructions.
Free amino acid content Free amino acids were extracted from natto as previously described (Nagai et al., 1994). Briefly, lyophilized natto samples were crushed to a powder. Amino acids were recovered in three rounds of extraction with 70% ethanol with vigorous shaking at 4 °C for 30 min followed by centrifugation at 9 700 × g at 4 °C for 5 min. The supernatant was subjected to HPLC analysis (Prominence 20 series; Shimadzu Co., Ltd.) employing the post-column o-phthalaldehyde derivative method as previously described (Kimura et al., 2004). Shim-pack Amino-Li column (Shimadzu Co., Ltd.), Li-type amino acids mobilephase kit, and amino acid reaction reagent kit (Shimadzu Co., Ltd.) were used as the mobile phase and reaction reagent, respectively. Standard solutions of amino acids (type AN-2 and B; FUJIFILM Wako Pure Chemical Co., Ltd.) were used for quantitative estimation of each amino acid by the absolute calibration curve method.
γ-PGA content Crude γ-PGA solution was prepared from natto and then purified as previously reported (Kubo et al., 2013). The γ-PGA sample was dissolved in water and hydrolyzed in 3 M hydrochloric acid in a block incubator at 110 °C for 4 h. After neutralization by sodium hydroxide, hydrolyzed samples were centrifuged at 15 000 × g at 4 °C for 5 min. Samples were then subjected to HPLC analysis to determine glutamic acid content as described in the previous section. Similarly, nonhydrolyzed samples were also subjected to HPLC analysis to determine the net glutamic acid content derived from γ-PGA. The γ-PGA content was determined from the glutamic acid content with a multiplier coefficient of 0.878.
Hardness of natto The hardness of natto grain was measured at 4 °C using a tensipresser (TTP-50BXII, Taketomo Electric, Inc.) (Sakamoto et. al., 2006). One grain of the natto sample was set on the stage of the tensipresser, and the maximum stress was measured when 80% compression of the whole grain was reached (circular plunger, 1 mm/s, ∅25 mm). A total of 40 grains were analyzed for each sample and measurement results for 20 grains are reported (upper and lower data values for 20 grains were eliminated) as previously described (Kubo et al., 2013).
Color The color of the natto surface was measured via colorimetry (SE-2000, Nippondenshoku Industries Co., Ltd.) using an attached optical fiber. For each sample, 10 grains were measured and evaluated based on values for L*, a*, b*, and C* (Hunt, 1977).
Statistical analysis Chemical and biological measurements were performed in triplicate and significant differences among values were determined by one-way analysis of variance and the Tukey-Kramer method (Tukey, 1953; Kramer, 1956).
Quality scoring for natto Ammonia content, free amino acid content, γ-PGA content, grain hardness, and L* and C* color values for stored natto were compared with those for fresh product (day 0). The quality score for natto (Qnatto) was defined by summing the absolute values of the relative differences.
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N, i and qN,i represent the storage time (days), the six proposed criteria and values for each of the six criteria, respectively. If the score was below 2.0, the stored natto was judged to be of acceptable quality.
B. subtilis viable count Bacterial number is an index to optically determine the extent of fermentation. Some animal experiments imply that there are health benefits associated with oral intake of B. subtilis cells (Murata et al., 1996a; Murata et al., 1996b). The initial bacterial number in our samples was 7.5 × 109 CFU/g, and B. subtilis cells continued to live during storage. At each tested storage temperature (5 °C, 10 °C, 20 °C), the bacterial number decreased to 7.4–9.0 × 108 CFU/g during storage, and no significant differences in viable cell count were observed among the samples (Table 1). Therefore, we concluded that under storage temperatures of 5–20 °C, viable B. subtilis cells survived in natto products for at least 20 days.
| Storage temperature | Storage period | Bacterial number | Ammmonium nitrogen content | Total free amino acid content | γ-PGA content |
|---|---|---|---|---|---|
| (°C) | (days) | (CFU/g) | (mg/100g) | (mg/100g d.w.) | (mg/100g) |
| - | 0 | 7.5 ± 0.6 × 109 a | 89.9 ± 1.0 i | 944 ± 5 g | 365.4 ± 10.7 bcd |
| 5 °C | 5 | 2.0 ± 0.1 × 109 b | 85.9 ± 0.9 i | 1,092 ± 22 fg | 329.4 ± 20.1 cde |
| 10 | 8.9 ± 1.5 × 108 c | 85.7 ± 9.1 i | 1,769 ± 9 ef | 301.7 ± 34.5 de | |
| 15 | 9.6 ± 2.4 × 108 c | 124.9 ± 1.3 fg | 2,448 ± 18 e | 346.5 ± 15.9 bcde | |
| 20 | 9.1 ± 1.1 × 108 c | 111.1 ± 2.5 gh | 2,443 ± 44 e | 292.7 ± 4.1 e | |
| 10 °C | 5 | 1.9 ± 0.2 × 109 b | 126.6 ± 1.6 f | 1,801 ± 21 e | 387.2 ± 21.6 abc |
| 10 | 9.4 ± 1.7 × 108 c | 109.2 ± 1.8 h | 2,377 ± 35 e | 358.1 ± 30.8 bcde | |
| 15 | 8.7 ± 2.1 × 108 c | 146.5 ± 0.2 e | 3,636 ± 115 d | 307.2 ± 29.3 de | |
| 20 | 7.4 ± 0.8 × 108 c | 186.8 ± 1.3 d | 3,691 ± 85 d | 309.4 ± 36.4 de | |
| 20 °C | 5 | 1.9 ± 0.1 × 109 b | 153.8 ± 3.2 e | 3,358 ± 242 d | 342.2 ± 21.8 bcde |
| 10 | 9.2 ± 0.4 × 108 c | 222.8 ± 0.8 c | 6,740 ± 148 c | 308.6 ± 22.9 de | |
| 15 | 1.1 ± 0.1 × 109 c | 283.4 ± 0.3 b | 9,242 ± 158 b | 450.1 ± 17.2 a | |
| 20 | 9.0 ± 2.3 × 108 c | 391.3 ± 7.7 a | 13,412 ± 748 a | 404.4 ± 24.0 ab | |
Natto manufactured by the conventional method with thermo-hygrostat (LH43-15p, Nagano Science Co. Ltd.) was stored at 5 °C–20 °C for 20 days and sampled every 5th day for analyses as described in the Materials and Methods. The results are expressed as mean ± SD values (n = 3). The values with different superscript letters in a column are significantly different (p < 0.05).
Ammonia nitrogen content Ammonia causes an irritating odor if it is over the sensory threshold level. Therefore, manufacturers and distributors need to control ammonia production from natto. Ammonia content in commercially available natto in Japan is reported to be 51–235 mg/100 g (wet weight) (Taira et al., 1987). In this study, the level of ammonia nitrogen was 89.9 ± 1.0 mg/100 g at day 0 (Table 1). Ammonia nitrogen constantly increased during the storage period at all tested temperatures (Table 1). At 5 °C, no significant difference was observed compared to the fresh sample until day 10, and a maximum ammonia nitrogen level (124 mg/100 g) was observed on day 15. At 10 °C and 20 °C, the ammonia nitrogen level reached 186.8 and 391.2 mg/100 g, respectively, on day 20 (Table 1).
The acceptable ammonia content of natto for consumers is reported to be 100–130 mg/100 g (Ohta, 1980). In this study, ammonia over the threshold level was present in samples stored at 10 °C for 15 days (146.5 mg/100 g) and 20 days (186.8 mg/g). Those stored at 20 °C had an unfavorable ammonia smell beginning on day 5 (153.8 mg/100 g).
Free amino acid content Free amino acids mainly arise in soybean protein through the action of proteases and peptidases, including AprE and YwaD produced by B. subtilis cells (Kada et al., 2013; Fundoiano-Hershcovitz et al., 2005; Liu et al., 2017). Although some amino acids like L-glutamic acid and L-serine are related to umami, others are sometimes associated with bitterness (Kawai et al., 2012). Therefore, an increased amino acid level potentially negatively affects quality. The free amino acid content in natto increased during storage, and it was significantly affected by temperature. Proteases/peptidases secreted outside cells by B. subtilis cells likely continued to generate free amino acids during the storage period.
The total free amino acid content per 100 g of dried natto is shown in Table 1. The amino acid content of the fresh (day 0) sample was 944 ± 5 mg/100 g. With the exception of the day 5 sample stored at 5 °C (1 092 ± 22 mg/100 g), other samples contained significantly higher amino acid content compared to the fresh sample (Table 1). For example, on day 20, the free amino acid content reached 2 443 ± 44, 3 691 ± 85, and 13 412 ± 748 mg/100 g at 5 °C, 10 °C, and 20 °C, respectively (Table 1). Abundant amino acids may contribute to the “umami” taste of natto. However, glutamic acid, glutamine, arginine, and proline are also sources of ammonia produced in natto (Takemura, 2014). Therefore, high amino acid content can additionally contribute to deterioration of natto quality due to ammonia odor. Changes in the free amino acid content mimicked changes observed in the ammonia nitrogen content (Table 1). The free amino acid content was highly correlated with that of ammonia nitrogen (correlation coefficient: 0.975). Major extracellular proteases of the natto bacterium do not function during storage at low temperature (Akimoto et al., 1993; Kashihara et al., 2001), which may account for the low levels of amino acids at 5 °C. Thus, temperature control is important to maintain the amino acid level.
γ-PGA content The γ-PGA content per 100 g is shown in Table 1. Compared to the fresh sample (365.4 ± 10.7 mg/100 g), the γ-PGA level decreased significantly when the sample was stored at 5 °C for 20 days (292.7 ± 4.1 mg/100 g; p < 0.05), and it increased significantly when the sample was stored at 20 °C for 15 days (450.1 ± 17.2 mg/100 g; p < 0.05). The gradual reduction of γ-PGA at 5 °C and 10 °C may imply that B. subtilis cells consumed γ-PGA as a nitrogen source to survive in the stationary phase (Kimura et al., 2004). In contrast, B. subtilis appeared to continue to synthesize γ-PGA at 20 °C. The nitrogen source was probably limited for continuous production of γ-PGA at low temperature (5 °C and 10 °C), which is far below the optimum temperature for extracellular exoenzymes. While the sticky texture of natto due to γ-PGA is considered to be a critical factor for quality of natto in Japan, less sticky natto was recently launched as an export product considering consumer acceptance overseas (Kubo and Nakagawa, 2018). Thus, the significance of the γ-PGA content may depend on the marketing strategy for natto.
Hardness of natto The hardness of the fresh sample was 8.4 ± 0.7 N. The hardness of natto tended to decrease during storage (Fig. 1). On day 5, natto stored at 5 °C and 10 °C showed less reduction of hardness than natto stored at 20 °C. However, natto rapidly softened during the next 5 days regardless of storage temperature (Fig. 1). At 20 days, the differences in hardness among samples with different storage temperature were less (4.3 ± 0.2 N at 5 °C, 4.4 ± 0.3 N at 10 °C, and 3.8 ± 0.2 N at 20 °C), and no significant differences were observed among them. These results suggest that softening of natto can not be prevented by temperature control.
Time-dependent hardness change of natto.
Natto was stored at 5 °C–20 °C for 20 days and sampled every 5th day for analyses. The hardness of natto was measured by Tensipresser as described in the Materials and Methods. The mean value of 20 natto gains stored at 5 °C (◆), 10 °C (■), and 20 °C (▲) are shown.
Color A high L* value (brightness) for natto is favorable, and discoloration gives a negative impression to consumers (Iwahashi et al., 2015). The L* value for the fresh product was 61.4 ± 2.2. For natto kept at 5 °C, no significant reduction of L* was observed for 15 days (Fig. 2). In contrast, a reduction of brightness was observed on day 5 for a storage temperature above 10 °C and the reduction in brightness was obvious for samples stored at 20 °C (Fig. 2). From day 10 to day 20, the L* values did not change significantly for all tested temperatures. The a* value (redness) for samples stored at 20 °C for more than 5 days and at 10 °C for more than 10 days exhibited a significant difference (p < 0.05) compared to the day 0 sample (Fig. 2). The b* value (yellowness) was also affected by temperature; at 5 °C, the b* value was 14.1 ± 1.5 on day 5, 13.5 ± 1.4 on day 10, 13.5 ± 0.9 on day 15, and 13.3 ± 1.5 on day 20. These values showed no significant differences compared to day 0 (13.0 ± 1.0; Fig. 2). At higher temperatures, the b* values decreased (8.1 ± 1.2 at 20 °C on day 20; Fig. 2). These results indicate that color changes in natto were largely due to browning which was typically affected by storage temperature. The Maillard reaction may be involved in this color change (Makino, 2007). By combining the a* value and b* value, the C* value (a*2+b*2)1/2 was determined. The C* value mean metric chroma and color became vivid with high C* value and dingy with low C* value. There were no significant differences in C* value at 5 °C, but a tendency for a reduction in C* value was seen at 10 °C and 20 °C along with longer storage period. This tendency was more pronounced at higher temperatures. We employed L* value and C* value as indicators of color change.
Time-dependent color changes of natto surface.
Natto was stored at 5 °C–20 °C for 20 days and sampled every 5th day for analyses as described in the Materials and Methods. The line plot indicates L* value (●) and bar charts indicate a* value, b* value, and C* value. The values are expressed as mean ± SD values (n = 10). Different letters indicate significant difference (p < 0.05).
Quality scoring for determination of freshness date We evaluated natto stored at different temperatures using six different parameters: viable B. subtilis cell count, ammonia nitrogen, free amino acids, γ-PGA, hardness, and color changes (L* value and C* value) for soybean grains. Comparing stored natto samples to fresh product enabled us to calculate quality scores for natto. The viable B. subtilis cell count was not significantly affected by the temperature conditions tested. Therefore, we did not use this value in the quality scoring. However, viable counts greater than 108 CFU/g may indicate completeness of fermentation.
The scoring results are shown in Table 2. Many natto manufacturers currently determine a best-before date for natto of approximately 10 days to provide a tasty product to consumers with the assumption that natto will be stored at or below 10 °C. In the present study, the quality score for natto stored at 10 °C for 10 days was 2.28. Considering that Japanese natto manufacturers deliver natto to market every day through cold chains, a score of 2.0 or below can be considered to ensure the quality of natto. After a 10-day storage at 5 °C, the quality score was 1.56. Thus, based on our results, 5 °C appears to be the recommended temperature for long-term storage and distribution of natto to avoid deterioration in quality.
| quality score1 | ||||
|---|---|---|---|---|
| Storage temperature | Storage period (days) | |||
| (°C) | 5 | 10 | 15 | 20 |
| 5 | 0.60 | 1.56 | 2.53 | 2.59 |
| 10 | 1.64 | 2.28 | 4.21 | 4.80 |
| 20 | 3.83 | 8.44 | 12.00 | 17.67 |
Natto samples stored at different temperatures were evaluated based on contents of ammonia nitrogen, free amino acids, gamma-polyglutamic acid (γ-PGA), hardness, and color changes (L* value and C* value) of soybean grains as described in the Materials and Methods.
.N: storage time (days), i: six proposed criteria (ammonia contents, free amino acid contents, γ-PGA contents, hardness of grain, L* and C* value of color), qN,i: each value for the 6 contents.
The six criteria for natto quality assessed in this study are basic parameters designed to rule out inferior products. We recommend standardization of assessment of natto quality for market expansion through cooperation among suppliers, distributers, and retail traders. Therefore, sensory evaluation for flavors and texture, and chemical parameters, such as moisture content and enzyme activity, are not included in the quality scoring parameters in this study. However, these parameters are thought to belong to a competitive area that will facilitate the market growth of natto. Exportation of natto has recently increased, and growth in overseas production of natto is projected. The quality scoring system as a key indicator of natto quality proposed in this study will help to grow the natto market worldwide.
We evaluated the quality of natto stored at various temperatures over time. Evaluation criteria included bacterial number, ammonia content, free amino acid content, γ-PGA content, grain hardness, and natto surface color. The bacterial number did not fluctuate over time, but as the storage period increased, the ammonia and free amino acid content increased while the hardness and L* value decreased. We scored the quality of stored natto samples using the six criteria and compared the quality scores to those for fresh natto. The scoring results suggested that storage conditions of 10 days at 5 °C and 5 days at 10 °C produced the highest quality natto. The quality score proposed in this study can help to expand the natto market worldwide and could serve as a key performance indicator useful for evaluating the distribution systems of natto manufacturers.
Acknowledgements This study was supported by a research promotion grant from the Japan Natto Cooperative Society Federation.
