A Natto Fermentation Composition and Its Application

Abstract

This invention provides a natto fermentation composition and its application, belonging to the field of natto fermentation technology. The natto fermentation composition provided by this invention includes *Lactobacillus casei*, *Kluyveromyces lactis*, and *Bacillus natto*. The natto fermentation composition provided by this invention significantly increases nattokinase activity while reducing ammonia odor, making the fermented natto more acceptable to consumers. The natto fermented using the natto fermentation composition and method of this invention has a nattokinase activity as high as 2620.43±4.05 U / mL, and the natto fermented by this invention has a low volatile basic nitrogen content and a high sensory evaluation.

Description

Technical Field

[0001] This invention belongs to the field of natto fermentation technology, and particularly relates to a natto fermentation composition and its application. Background Technology

[0002] Natto, a fermented soybean product made from soybeans using Bacillus subtilis (Natto bacteria), is sticky, has a strong odor, and a slightly sweet taste. It not only retains the nutritional value of soybeans, is rich in vitamin K2, and improves protein digestibility and absorption, but more importantly, the fermentation process produces various physiologically active substances such as nattokinase, which has health benefits such as dissolving fibrin and regulating other physiological functions. Natto originated in China, but Japanese scholars later conducted in-depth research and discovered nattokinase and Bacillus subtilis. The patented natto fermentation strains have long been controlled by Japan, which has greatly limited the development of my country's natto food industry. For example, commercially available natto in my country has a fishy and pungent taste, making it unacceptable to consumers. Therefore, developing fermentation strains and methods that can produce flavor compounds, improve nutritional value, and effectively reduce the unpleasant odor of natto has good economic benefits and broad development prospects. Summary of the Invention

[0003] In view of this, the purpose of the present invention is to provide a natto fermentation composition. Natto fermentation using the natto fermentation composition of the present invention can significantly increase the activity of nattokinase in the fermentation product while significantly reducing the content of volatile basic nitrogen, reducing ammonia odor, and improving the sensory evaluation of natto products.

[0004] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0005] The present invention provides a natto fermentation composition comprising Lactobacillus casei, Kluyveromyces lactis, and Bacillus natto.

[0006] Preferably, the volume ratio of Bacillus natto, Lactobacillus casei, and Kluyveromycin is 1-10:1:1.

[0007] Preferably, the Bacillus subtilis natto is Bacillus subtilis AS20.

[0008] The present invention also provides the application of the above composition in natto fermentation.

[0009] The present invention also provides a method for fermenting natto, wherein the above-mentioned natto fermentation composition is inoculated onto soybeans and fermented.

[0010] Preferably, the process includes the following steps: first, inoculating soybeans with the above-mentioned Bacillus natto for fermentation for 8-10 hours, then inoculating soybeans with the above-mentioned Lactobacillus casei for fermentation for 8-10 hours, and finally inoculating soybeans with the above-mentioned Kluyveromyces lactis for fermentation for 8-10 hours.

[0011] Preferably, the fermentation temperature is 33-39℃.

[0012] Preferably, the inoculation amount of Bacillus natto is 5%-10% of the volume of the fermentation broth.

[0013] Preferably, the inoculation ratio of Bacillus natto, Lactobacillus casei, and Kluyveromyces lactis is 1-10:1:1.

[0014] The beneficial effects of this invention are:

[0015] The natto fermentation composition provided by this invention significantly increases nattokinase activity while reducing ammonia odor, making the fermented natto more acceptable to consumers. The natto fermented using this invention's composition and method exhibits a nattokinase activity as high as 2620.43±4.05 U / mL, higher than commercially available natto (generally between 1137.15 U / mL and 1222.727 U / mL). Furthermore, the natto fermented using this invention has a lower volatile basic nitrogen content and a higher sensory evaluation. Its volatile basic nitrogen content is 45.62±1.32 mg / 100g, far lower than commercially available natto (generally above 150 mg / 100g).

[0016] The Bacillus subtilis AS20 fermentation method provided by this invention has high nattokinase activity, and its application in natto fermentation can significantly improve the nutritional and health benefits of natto. Attached Figure Description

[0017] Figure 1 Results of ARTP mutagenesis lethality and positive mutation rate of Bacillus natto S6;

[0018] Figure 2 To measure the nattokinase activity of different strains;

[0019] Figure 3 The results indicate the genetic stability of the mutant strain;

[0020] Figure 4 The effect of mixed fermentation of Streptococcus thermophilus and AS20 on the viable count of Bacillus natto;

[0021] Figure 5 The effect of mixed fermentation of Lactobacillus plantarum and AS20 on the viable count of Bacillus natto;

[0022] Figure 6 The effect of mixed fermentation of Lactobacillus casei and AS20 on the viable count of Bacillus natto;

[0023] Figure 7 The effect of mixed fermentation of SR and AS20 on the viable count of lactic acid bacteria;

[0024] Figure 8 The effect of mixed fermentation of GL and AS20 on the viable count of lactic acid bacteria;

[0025] Figure 9 The effect of mixed fermentation of ZW and AS20 on the viable count of lactic acid bacteria;

[0026] Figure 10 To investigate the effects of different fermentation processes on the viable count of Bacillus subtilis AS20 in fermented natto;

[0027] Figure 11 The effects of different fermentation processes on volatile basic nitrogen in fermented natto;

[0028] Figure 12 The effects of different fermentation processes on the nattokinase activity of fermented natto;

[0029] Figure 13 The effect of different fermentation processes on the sensory scores of fermented natto;

[0030] Figure 14 The effect of different inoculum amounts of the three bacteria on the volatile basic nitrogen content;

[0031] Figure 15 The effect of different inoculum amounts of three bacteria on nattokinase activity;

[0032] Figure 16 The effect of different inoculum amounts of three bacteria on the sensory evaluation of natto;

[0033] Figure 17 The effect of different fermentation times on the content of volatile basic nitrogen;

[0034] Figure 18 The effect of different fermentation times on nattokinase activity;

[0035] Figure 19 The effect of different fermentation times on the sensory evaluation of natto;

[0036] Figure 20 The effect of different fermentation temperatures on the content of volatile basic nitrogen;

[0037] Figure 21 The effect of different fermentation temperatures on nattokinase activity;

[0038] Figure 22 The effect of different fermentation temperatures on the sensory evaluation of natto. Detailed Implementation

[0039] The present invention provides a natto fermentation composition comprising Lactobacillus casei, Kluyveromyces lactis, and Bacillus natto.

[0040] This invention does not specifically limit the sources of *Lactobacillus casei*, *Kluyveromyces lactis*, and *Bacillus natto*, and commercially available products conventional in the art can be used. In this invention, the volume ratio of *Bacillus natto*, *Lactobacillus casei*, and *Kluyveromyces lactis* is preferably 1-10:1:1, more preferably 5:1:1. In this invention, the *Bacillus natto* is preferably *Bacillus subtilis* AS20, which is obtained by ARTP mutagenesis starting with *Bacillus natto* S6. Natto fermented with *Bacillus natto* AS20 of this invention exhibits high nattokinase activity. The *Bacillus subtilis* AS20 of this invention can be obtained from the website https: / / zysw.snut.edu.cn / info / 1134 / 1980.htm and is guaranteed to be available to the public within twenty years from the date of application.

[0041] The present invention also provides the application of the above composition in natto fermentation.

[0042] The present invention also provides a method for fermenting natto, wherein the above-mentioned natto fermentation composition is inoculated onto soybeans and fermented.

[0043] In the natto fermentation method of the present invention, the preferred steps include: first inoculating soybeans with the above-mentioned Bacillus natto for fermentation for 8-10 hours, then inoculating soybeans with the above-mentioned Lactobacillus casei for fermentation for 8-10 hours, and finally inoculating soybeans with the above-mentioned Kluyveromyces lactis for fermentation for 8-10 hours.

[0044] This invention does not specifically limit the source of soybeans; any commercially available product in the field is acceptable. In this invention, the fermentation temperature is preferably 33-39℃, more preferably 36℃. The fermentation time after inoculation with *Bacillus natto* is preferably 9 hours, followed by inoculation with *Lactobacillus casei*. After inoculation with *Lactobacillus casei*, the fermentation time is preferably 9 hours, followed by inoculation with *Kluyveromyces lactis*. The fermentation process is then complete. In this invention, the inoculation amount of *Bacillus natto*, based on the volume of the fermentation broth, is preferably 5%-10%. The inoculation ratio of *Bacillus natto*, *Lactobacillus casei*, and *Kluyveromyces lactis* is preferably 1-10:1:1, more preferably 5:1:1.

[0045] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0046] Unless otherwise specified, the following embodiments are all conventional methods.

[0047] Unless otherwise specified, all materials and reagents used in the following examples are commercially available. The data analysis methods used in the following examples are as follows: SPSS 20.0 statistical software was used for significance analysis of the experimental data, and the results are expressed as mean ± standard deviation (AVE ± SD). Excel 2016 and other relevant software were used for image and chart processing.

[0048] Example 1

[0049] ARTP mutagenesis: The metal slide carried by the ARTP breeder was moved to the clean bench, and 10 μL of the natto strain S6 suspension from our laboratory was spread on the metal slide. The plate was then moved to the operating chamber of the ARTP mutagenesis system. According to the operating procedure of the ARTP bio-breeder, it was set to 100W, air flow rate of 10 SLM, and the distance between the slide and the air port was 3 mm. The mutagenesis treatment time was set to 0, 20s, 40s, 60s, 80s, 100s, 120s, 140s, and 160s.

[0050] After mutagenesis, the metal slides were eluted with sterile physiological saline to generate a new bacterial suspension. The suspension was agitated thoroughly and then serially diluted and plated onto casein plates. Each treatment was repeated three times. The plates were incubated upside down at 37°C for 24 hours. Colony growth was observed, and colony counts were performed. The lethality rate of the strain was calculated using the following formula. In calculating the positive mutation rate, systematic errors were considered. Mutant strains were selected, and those whose enzyme activity was 10% higher than the control group after the first shake-flask fermentation were considered positive mutant strains. Results are as follows... Figure 1 As shown, when the treatment time reaches 160s, the lethality reaches its maximum of 99%, and the positive mutation rate is relatively low at 3.6%. When the mutagenesis treatment time is 100s, the lethality is 87%, and the highest positive mutation rate is 9%. A high positive mutation rate is beneficial for the screening of bacterial strains, so 100s was chosen as the mutagenesis treatment time.

[0051] Lethality (%) = (Control group - Total number of colonies in the mutagenized group) / Total number of colonies in the control group × 100

[0052] Positive mutation rate (%) = (Number of colonies of positive mutant strain / Total number of colonies grown after treatment) × 100

[0053] Typically, when the lethality rate is around 90%, the strain is more likely to exhibit high-yield mutations, but the reproducibility at this mutation rate is also relatively poor. Therefore, strains with a lethality rate of 90% are selected for mutation.

[0054] Screening of Mutagenic Strains: After appropriate dilution, the ARTP-mutated bacterial suspension was spread onto casein plates and incubated upside down at 37°C for 24 hours. Suspected mutant strains were selected by comparing the colony diameter with the diameter of the proteolytic zone on the casein plate. These mutant strains were then verified by shake-flask fermentation. Seed culture of the strain in the logarithmic phase (incubated at 37°C in a shaker at 180 rpm for 7-9 hours until the strain reaches the logarithmic phase, the peak period for extracellular enzyme production in Bacillus) was inoculated at a rate of 5% into 250 mL Erlenmeyer flasks containing 50 mL of fermentation medium. Fermentation was carried out at 37°C and 180 rpm for 24 hours, followed by secondary screening based on nattokinase activity. Results: After four rounds of ARTP mutagenesis, 304 strains with clear transparent zones were obtained from the initial casein screening plates of Bacillus natto S6. 34 single colonies with a proteolytic zone diameter larger than the colony diameter were selected for shake-flask fermentation verification. The starting strain was Bacillus natto S6, which was previously screened in the laboratory. Mutant strains were designated with the initial "A" of ARTP, and the serial numbers were the order markers of the bacteria growing on each mutagenesis plate. A total of four positive mutant strains were obtained, with a positive mutation rate of 11.76%. Among them, AS20 exhibited the highest nattokinase activity, at 2461.72 ± 6.80 U / mL, a 15.7% increase compared to the original strain (2074.16 U / mL). The nattokinase activity results of these four strains are as follows: Figure 2 As shown, there was no significant difference in the effect of different strains on nattokinase activity (P>0.05).

[0055] Determination of genetic stability of the mutagenic strain: The selected mutagenic strain AS20 with high nattokinase activity was agar-agar in test tubes and stored at 4°C. During subculturing and activation, a loopful was picked from the agar slant and streaked onto a fresh agar slant. The agar slant was then incubated at 37°C for 24 hours. This process was repeated 10 times, with shake-flask fermentation performed every two generations to determine the nattokinase activity. Results are as follows: Figure 3 As shown, the nattokinase activity of the mutant strain AS20 remained relatively stable after 10 generations of passage. It exhibited high nattokinase activity in the 4th generation and maintained this level without significant decrease in the 10th generation. Statistical analysis (F = 0.25, P = 0.32) demonstrates that this mutant strain possesses good genetic stability.

[0056] Commercially available soybeans with plump and intact grains were selected as the test material. The soybeans were washed with clean water and soaked in distilled water overnight at room temperature to ensure they were fully dried. The drained soybean grains were placed in 3.5cm thick fermentation cups of the same size and sterilized at 121℃ for 20 minutes. Under aseptic conditions, they were allowed to cool naturally to room temperature. After incubation at 37℃ and 170 rpm for 16 hours, the mixture was shake-activated. The activated Bacillus subtilis AS20 and the starting strain Bacillus subtilis S6 were sprayed onto the soybean surface at a 5% inoculum volume (based on the fermentation liquid volume). The mixture was stirred thoroughly, sealed with a breathable membrane, and fermented at 37℃ for 24 hours. After fermentation, the mixture was placed in a 4℃ refrigerator for 24 hours for post-fermentation to complete the natto production. The volatile basic nitrogen content in the two types of natto was determined by semi-micro nitrogen determination method, the nattokinase activity in the two types of natto was determined by Folin-phenol method, and the sensory evaluation of the two types of natto was carried out. The sensory evaluation criteria are shown in Table 1. The natto was evaluated by blind evaluation mode, and the results are shown in Table 2.

[0057] Table 1 Sensory Evaluation Criteria

[0058]

[0059] Table 2 Results of various indicators before and after strain mutagenesis

[0060]

[0061] Example 2

[0062] A natto fermentation composition comprises Lactobacillus casei (purchased from Shanghai Luwei Technology Co., Ltd., LWCC3002), Kluyveromyces lactis (purchased from Ningbo Mingzhou Biotechnology Co., Ltd., B336121) and Bacillus subtilis AS20 natto obtained by mutagenesis in Example 1, wherein the volume ratio of Bacillus subtilis AS20 natto, Lactobacillus casei and Kluyveromyces lactis is 5:1:1.

[0063] Example 3

[0064] The difference from Example 2 is that the volume ratio of Bacillus subtilis AS20, Lactobacillus casei and Kluyveromyces lactis is 1:1:1.

[0065] Example 4

[0066] The difference from Example 2 is that the volume ratio of Bacillus subtilis AS20, Lactobacillus casei and Kluyveromyces lactis is 10:1:1.

[0067] Example 5

[0068] The Bacillus subtilis AS20 obtained in Example 1 was inoculated into NBP liquid medium and cultured at 37°C and 170 r / min for 16 h for shake-flask activation. The shake-flask activated Bacillus subtilis AS20 was then mixed with Streptococcus thermophilus (purchased from Shanghai Luwei Technology Co., Ltd., denoted as SR), Lactobacillus plantarum (purchased from Shanghai Luwei Technology Co., Ltd., denoted as ZW), and Lactobacillus casei (purchased from Shanghai Luwei Technology Co., Ltd., denoted as GL) respectively (the mixing ratio of any two of the above bacteria was 1:1). After that, 5% of the inoculum (based on the volume of the fermentation broth, 5% of the volume) was sprayed onto the surface of soybeans. Different fermentation times were designed to compare the effects of different fermentation times on the viable counts of Bacillus subtilis and lactic acid bacteria in different groups of mixed fermented natto (using the plate dilution and coating count method).

[0069] The results are as follows Figures 4-9 As shown, the mixed fermentation of Lactobacillus casei and Bacillus natto exhibited the best growth, with a viable count of (11.71±0.42)×10⁻⁶ Bacillus natto. 6 CFU / mL. Statistical analysis showed that time had a significant effect on the viable bacterial count (P < 0.05). *Lactobacillus thermophilus*, *Lactobacillus plantarum*, and *Lactobacillus casei* all showed good growth, with *Streptococcus thermophilus* and *Lactobacillus plantarum* showing poorer results, while *Lactobacillus casei* showed the best growth at (9.23 ± 0.05) × 10⁻⁶ CFU / mL. 6 CFU / mL. Statistical analysis showed that time had a significant effect on the viable count of the three lactic acid bacteria (P < 0.05).

[0070] according to Figures 4-9 The optimal fermentation process was used to complete the production of natto. The volatile basic nitrogen content (detection method as in Example 1), nattokinase activity (detection method as in Example 1), and sensory scores of fermented natto were compared among different groups. The results are shown in Table 3. In Table 3, the AS20 group represents the data obtained after fermenting natto alone using the mutagenized Bacillus subtilis AS20 described in Example 1, following the method in Example 1. When Lactobacillus casei and Bacillus natto were mixed and fermented, the ammonia odor was significantly reduced, and the volatile basic nitrogen content was 92.43±3.08 mg / 100g. Different strain combinations showed significant differences in their effects on the volatile basic nitrogen content of natto (P<0.05). After mixed fermentation of Lactobacillus casei and Bacillus natto, the enzyme activity not only did not decrease but also increased to 2134.12±4.05 U / mL. Different strain combinations showed significant differences in their effects on nattokinase activity (P<0.05).

[0071] Table 3 Results of fermentation of Bacillus subtilis AS20 with different lactic acid bacteria in natto

[0072]

[0073] Example 6

[0074] A method for fermenting natto: First, inoculate soybeans with Bacillus subtilis AS20 (inoculation amount 5%, based on the volume of the fermentation liquid, inoculating 5% of the volume) and ferment at 37°C for 8 hours. Then, inoculate with Lactobacillus casei and ferment at 37°C for 8 hours. Finally, inoculate with Kluyveromyces lactis and ferment at 37°C for 8 hours (the inoculation amount of Lactobacillus casei and Kluyveromyces lactis is the same as that of Bacillus subtilis AS20).

[0075] Comparative Example 1

[0076] The difference from Example 6 is that the three bacteria were inoculated onto soybeans at the same time and fermented for 24 hours, while the rest were the same as in Example 6.

[0077] Comparative Example 2

[0078] The difference from Example 6 is that Bacillus subtilis AS20 was first inoculated and fermented for 8 hours, followed by inoculation with lactic acid bacteria and Kluyveromyces lactis and fermented for 16 hours. The rest is the same as Example 6.

[0079] Comparative Example 3

[0080] The difference from Example 6 is that Bacillus subtilis AS20 was first inoculated and fermented for 8 hours, then Kluyveromyces lactis was inoculated and fermented for 8 hours, and finally lactic acid bacteria were inoculated and fermented for 8 hours. The rest is the same as Example 6.

[0081] The effects of Example 6 and Comparative Examples 1-3 on the viable count of Bacillus subtilis in natto after fermentation, as well as nattokinase activity, volatile basic nitrogen content, and sensory scores of natto from different groups, were determined. The specific measurement methods and sensory evaluation methods were the same as in Examples 1 and 5. Example 6 was designated as Group 3, Comparative Example 1 as Group 1, Comparative Example 2 as Group 2, and Comparative Example 3 as Group 4. The results are as follows: Figure 10-13 As shown.

[0082] Depend on Figure 10 It can be seen that in Group 1, simultaneous inoculation of the three bacteria significantly inhibited the growth of Bacillus natto due to the rapid growth of lactic acid bacteria and yeast. At the end of fermentation, the viable Bacillus natto count was only (4.26±0.92)×10⁻⁶. 6 CFU / mL. In group 2, Bacillus natto was used for fermentation first, followed by co-fermentation with lactic acid bacteria and yeast. The co-fermentation with lactic acid bacteria and yeast may have had some influence, resulting in a less than ideal outcome; the viable cell count was only (5.61±0.24)×10⁻⁶. 6 The CFU / mL level was lower than that of groups 3 and 4.

[0083] Depend on Figure 11It can be seen that the highest volatile basic nitrogen content was observed in Group 1, where all three bacteria were inoculated simultaneously. This may be because the growth of lactic acid bacteria and yeast in this group was poor, failing to effectively improve the unpleasant flavor of natto. The latter three groups, due to the acid-producing and aroma-generating effects of lactic acid bacteria and yeast, produced fermented natto with a certain aroma, resulting in lower volatile basic nitrogen content compared to Group 1. Group 3 exhibited the lowest volatile basic nitrogen content, indicating the best effect. Statistical analysis showed a significant difference in the impact of different fermentation processes on volatile basic nitrogen content (P<0.05).

[0084] Depend on Figure 12 It can be seen that the nattokinase activity in group 3 was the highest. This may be because the fermentation products of Bacillus natto promote the growth of lactic acid bacteria and yeast, and are also more conducive to the growth of Bacillus natto. Statistical analysis shows that there are significant differences in the effects of different fermentation processes on the volatile basic nitrogen content (P<0.05).

[0085] Depend on Figure 13 It can be seen that the sensory score of Group 1 was the lowest at 58±1.42 points. This may be because the viable counts of lactic acid bacteria and yeast in Group 1 were very low, and their growth was also poor, making it difficult to effectively improve the unpleasant flavor of natto. The latter three groups, due to the acid-producing and aroma-generating effects of lactic acid bacteria and yeast, produced fermented natto with a good sour and fragrant flavor, a softer and more glutinous texture, and longer stringy consistency, resulting in higher sensory scores of 64.03±0.25 points, 62.15±0.31 points, and 61.04±0.68 points, respectively. Statistical analysis showed that the fermentation process had no significant difference in enzyme activity (P>0.05). Considering all factors, Group 3 had the highest viable count of Bacillus natto and the highest nattokinase, and its sensory score was also significantly higher than that of Groups 2 and 4, and much lower than that of Group 1.

[0086] Example 7

[0087] A method for fermenting natto: First, inoculate soybeans with Bacillus subtilis AS20 (inoculation amount is 5% based on the volume of natto fermentation liquid before fermentation) and ferment at 36℃ for 8 hours. Then, inoculate with Lactobacillus casei (inoculation amount is the same as Bacillus subtilis AS20) and ferment at 36℃ for 8 hours. Finally, inoculate with Kluyveromycin Lactobacillus (inoculation amount is the same as Bacillus subtilis AS20) and ferment at 36℃ for 8 hours. After fermentation, allow for 24 hours of post-ripening.

[0088] Example 8

[0089] The difference from Example 7 is that the inoculation ratios of Bacillus subtilis AS20, Lactobacillus casei, and Kluyveromyces lactis were adjusted to 10:1:1, 5:1:1, 1:1:5, 1:1:10, 1:5:1, and 1:10:1, respectively; all other ratios were the same as in Example 7. The effects of different bacterial ratios on volatile basic nitrogen, nattokinase, and sensory evaluation in fermented natto were compared between Examples 7 and 8. The specific detection and evaluation methods were the same as in Example 1, with three replicates per group, and the average value was taken. Figure 14-16 As shown.

[0090] Depend on Figure 14 It can be seen that, with other fermentation conditions remaining constant, changing only the inoculum ratio has a significant impact on the volatile basic nitrogen content of fermented natto. The lowest volatile basic nitrogen content was reached at a ratio of 10:1:1, which was 52.71 ± 0.62 mg / 100g. Statistical analysis shows that different inoculum amounts have a significant effect on the viable count of Bacillus natto (P < 0.05).

[0091] Depend on Figure 15 It can be seen that, with other fermentation conditions remaining constant, changing only the inoculum ratio has a significant impact on the nattokinase activity of fermented natto. The nattokinase activity reached its highest value of 2533.08 ± 4.26 U / mL when the inoculum ratio was 5:1:1. Statistical analysis showed that there was no significant difference in the effect of different inoculum ratios on nattokinase activity (P > 0.05).

[0092] Depend on Figure 16 It can be seen that, with other fermentation conditions remaining constant, changing only the ratio of microbial strains has a significant impact on the sensory evaluation of fermented natto. The sensory score reaches its highest value at a ratio of 5:1:1.

[0093] Taking all factors into consideration, when the ratio of microbial strains is 5:1:1, the content of volatile basic nitrogen in natto is low, while the nattokinase activity and sensory scores also reach their highest values. Therefore, the optimal microbial strain ratio for 3-strain fermentation is 5:1:1.

[0094] Example 9

[0095] The difference from Example 7 lies in the change of fermentation time. The fermentation times of the three bacteria were simultaneously modified to 9h, 10h, 11h, and 12h (i.e., the total fermentation times were 27h, 30h, 33h, and 36h, respectively). Specifically, when the total fermentation time was 27h, the specific method was to first inoculate soybeans with Bacillus subtilis AS20 for 9h, then inoculate with Lactobacillus casei for 9h, and finally inoculate with Kluyveromyces lactis for 9h, and so on). All other aspects were the same as in Example 7. The effects of different fermentation times in Examples 7 and 9 on volatile basic nitrogen, nattokinase, and sensory evaluation in fermented natto were compared. The specific detection and evaluation methods were the same as in Example 1. Each group of experiments was repeated in triplicate, and the results were averaged. Figure 17-19 As shown.

[0096] As fermentation time increased, the content of volatile basic nitrogen also increased, reaching its lowest point after 24 hours of fermentation. Statistical analysis showed that different fermentation times had a significant impact on the volatile basic nitrogen content (P < 0.05). Nattokinase activity increased continuously between 24 and 27 hours of fermentation, then decreased after 30 hours. Statistical analysis also showed a significant impact of different fermentation times on nattokinase activity (P < 0.05). The highest sensory score was observed in natto fermented at 27 hours. Considering all factors, the optimal fermentation time for the three-strain fermentation was 27 hours, as the volatile basic nitrogen content was relatively low, while nattokinase activity and sensory scores were highest.

[0097] Example 10

[0098] The difference from Example 7 lies in the change of fermentation temperature. The fermentation temperatures of the three bacteria were simultaneously modified to 30℃, 33℃, 39℃, and 42℃, respectively; all other aspects remained the same as in Example 7. The effects of different fermentation temperatures on volatile basic nitrogen, nattokinase, and sensory evaluation in fermented natto were compared between Examples 7 and 10. Specific detection and evaluation methods were the same as in Example 1, with three replicates for each group of experiments, and the results were averaged. Figure 20-22 As shown.

[0099] At temperatures between 30℃ and 36℃, the volatile basic nitrogen content is relatively low. Statistical analysis shows that there is no significant difference in the effect of different fermentation temperatures on the volatile basic nitrogen content (P > 0.05). Between 30℃ and 36℃, enzyme activity increases continuously with increasing fermentation temperature, but after 36℃, enzyme activity decreases continuously with increasing fermentation temperature. Statistical analysis shows that there is a significant difference in the effect of different fermentation temperatures on nattokinase activity (P < 0.05). The sensory score of natto reaches its maximum at 36℃. Statistical analysis shows that there is a significant difference in the effect of different fermentation temperatures on the sensory score (P < 0.05). Considering all factors, it can be concluded that the volatile basic nitrogen content is relatively low, while the nattokinase activity and sensory score are highest at 37℃. Therefore, the optimal fermentation temperature for the three-strain fermentation is 37℃.

[0100] Example 11

[0101] Based on the natto fermentation method in Example 7, an orthogonal experiment was conducted with three factors and three levels: strain ratio, fermentation time, and fermentation temperature, as shown in Table 4.

[0102] Table 4 Orthogonal Experimental Design

[0103]

[0104] The volatile basic nitrogen, nattokinase, and sensory evaluation of natto in different groups were compared. The specific detection and evaluation methods were the same as in Example 1, as shown in Table 5.

[0105] By comparing R-values, the order of influence of each factor on each evaluation index can be determined; by comparing K-values, the optimal combination of factors can be determined. The results analysis shows that: the order of influence on volatile basic nitrogen content is A > B > C, and the optimal combination is A2B1C1, i.e., a microbial ratio of 5:1:1, a fermentation time of 24 h, and a fermentation temperature of 36℃. The order of influence on nattokinase activity is A > B > C, and the optimal combination is A2B1C1, i.e., a microbial ratio of 5:1:1, a fermentation time of 24 h, and a fermentation temperature of 36℃. The order of influence on sensory scores is A > B > C, and the optimal combination is A2B1C2, i.e., a microbial ratio of 5:1:1, a fermentation time of 24 h, and a fermentation temperature of 39℃.

[0106] Factors A and B have the same effect on the strain ratio, fermentation time, and fermentation temperature: A2B1. Factor C has the third largest effect on all three factors. When using volatile basic nitrogen content and nattokinase activity as evaluation criteria, C1 should be selected; when using sensory scores, C2 should be selected. Therefore, the choice should be between C1 and C2. Although the sensory score is slightly lower than C2, the volatile basic nitrogen content is significantly lower, and the enzyme activity is significantly higher. Therefore, factor C should be C1. Thus, the optimal combination of the three factors is A2B1C1, i.e., a strain ratio of 5:1:1, a fermentation time of 24 hours, and a fermentation temperature of 36℃.

[0107] Verification experiments were conducted, with three replicates performed and the average results taken. The volatile basic nitrogen content of the natto fermented with the three strains was 45.62±2.07 mg / 100g, the nattokinase activity was 2620.43±4.19 U / mL, and the sensory score was 59.37±1.24. Compared to single-strain fermentation (an additional experiment using AS20 as a control was conducted to replicate the optimal fermentation conditions), the volatile basic nitrogen content decreased by 72.19%, the nattokinase activity increased by 24.59%, and the sensory score increased by 28.26%.

[0108] Table 5 Results of the orthogonal experiment

[0109]

[0110] Example 12

[0111] A natto fermentation composition comprising Lactobacillus casei (purchased from Shanghai Luwei Technology Co., Ltd., LWCC3002), Kluyveromyces lactis (purchased from Ningbo Mingzhou Biotechnology Co., Ltd., B336121) and Bacillus natto (purchased from Ningbo Taisto Biotechnology, TS287475), wherein the volume ratio of Bacillus natto, Lactobacillus casei and Kluyveromyces lactis is 5:1:1.

[0112] Example 13

[0113] A method for fermenting natto: Soybeans are fermented using the natto fermentation composition described in Example 12. The specific steps are as follows: First, Bacillus natto is inoculated onto the soybeans (inoculation amount is 5% based on the volume of the natto fermentation liquid before fermentation) and fermented at 36°C for 8 hours. Then, Lactobacillus casei is inoculated (inoculation amount is the same as Bacillus natto AS20) and fermented at 36°C for 8 hours. Finally, Kluyveromyces lactis is inoculated (inoculation amount is the same as Bacillus natto AS20) and fermented at 36°C for 8 hours. After fermentation, it is post-ripened for 24 hours to obtain fermented natto. The volatile basic nitrogen content, nattokinase, and sensory evaluation of the obtained fermented natto are detected using the same methods as in Example 1. The results show: nattokinase activity 2103.16±1.83 U / mL; volatile basic nitrogen content 121.05±3.20 mg / 100g; sensory evaluation score 47.29±0.74 points.

[0114] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A natto fermentation composition, characterized by, The composition is composed of Lactobacillus casei, Kluyveromyces lactis and Bacillus natto; The volume ratio of Bacillus natto, Lactobacillus casei and Kluyveromyces lactis is 1-10:1:1; The Bacillus natto is Bacillus subtilis natto AS20.

2. The composition of claim 1 is used in natto fermentation.

3. A method for fermenting natto, characterized by, The natto fermentation composition of claim 1 is inoculated on soybeans for fermentation.

4. The method of claim 3, wherein the fermentation is performed at a temperature of 30 to 40°C. The method comprises the following steps: inoculating the Bacillus natto on soybeans for 8-10 hours, inoculating the Lactobacillus casei for 8-10 hours, and inoculating the Kluyveromyces lactis for 8-10 hours.

5. The method of claim 3, wherein, The fermentation temperature is 33-39℃.

6. The method of claim 4, wherein, The inoculation amount of the Bacillus natto is 5%-10% of the volume of the fermentation liquid.

7. The method of claim 4, wherein, The inoculation amount ratio of the Bacillus natto, Lactobacillus casei and Kluyveromyces lactis is 1-10:1:1.

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