Composite fermentation inoculant and application thereof

Abstract

The invention relates to the technical field of microorganisms, in particular to a staphylococcus carnosus ZF626 and pediococcus acidilactici ZF627 composite fermentation inoculant and application thereof. The composite fermentation inoculant provided by the invention has high salt resistance and high ester yield, is used for fermentation of fermented soya beans, and can significantly increase the content of various key volatile compounds in the fermented soya beans, including the total content of flavor substances such as alcohols, esters, phenols, acids and the like, especially the content of key flavor compounds such as isoamyl alcohol, ethyl acetate, gamma-caprolactone, guaiacol and the like. The compound fermentation inoculant is used for fermenting the fermented soya beans, so that the flavor, taste and aroma coordination of the fermented soya beans can be remarkably improved, the overall aroma and flavor of the fermented soya beans are improved, the flavor and taste of the fermented soya beans are richer, more complex, layered and recognizable, and the overall flavor and quality of the fermented soya beans are remarkably improved.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a composite fermentation bacterial agent of a Staphylococcus carnosus ZF626 and a Pediococcus acidilactici ZF627 and an application thereof. Background Art

[0002] As a traditional fermented soybean product that is deeply loved by consumers, fermented black beans are famous for their unique flavor, rich nutritional value and the characteristics of being both a medicine and food. The production process of fermented black beans is actually to use various enzyme systems such as proteases and cellulases secreted by microorganisms during the koji making process to decompose macromolecules such as proteins in soybeans to generate small molecular peptides, amino acids and monosaccharides. These flavor precursors undergo a series of biochemical reactions in the later fermentation process to form the color, aroma and taste of fermented black beans. Traditional fermented black beans are generally fermented under natural conditions. The differences in geographical environment and fermentation microorganisms lead to differences in the quality of the finished black beans, which seriously restricts the development of the industry. In order to achieve industrialization, it is necessary to study the pure fermentation process of black beans. In addition, the traditional fermented black beans have a unique and rich flavor. With the industrial production of black beans, its flavor is far inferior to that of naturally fermented black beans. Therefore, the improvement of the flavor of pure fermented black beans is also the focus of current research.

[0003] At present, there are many studies on fermentation microorganisms of fermented black beans, but they are mainly focused on molds and yeasts, and there are relatively few studies on lactic acid bacteria. CN 104996931A discloses a method for making flavored black beans, which uses Bacillus subtilis and Lactobacillus plantarum to mix and ferment in steps and combine with low-temperature post-fermentation to produce black beans with good flavor. This method can increase the concentration of diacetyl and 2,3-butanediol in black beans, but the effect of improving the flavor of black beans is limited. Patent CN117678701A discloses a rapid fermentation method for reducing the salinity of black beans and improving the flavor. The black beans are fermented by inoculating Pediococcus acidilactici and Zygosaccharomyces rouxii, and the fermented bean embryos are compounded with spices, liquor extract and licorice extract to obtain a black bean product with a strong sauce and soy sauce flavor. However, this method has a complex production process and high production cost, and is not suitable for large-scale production. Patent CN117981843A discloses a composite fermentation agent and its application in light fermented black beans. The composite fermentation agent includes: brewer's yeast, plant lactic acid bacteria, Foshan Fengliao's light fermented black beans product, aralia kiwifruit juice, thiolated codonopsis polysaccharide, sodium citrate, and the flavor, taste and nutritional activity of the inoculated and fermented light fermented black beans are significantly improved. However, the formula of the composite fermentation agent is relatively complex, which increases the difficulty of large-scale production. Summary of the invention

[0004] Based on the above technical problems, the main purpose of the present invention is to overcome the shortcomings of the above-mentioned background technology and provide a composite fermentation agent comprising Staphylococcus carnosus ZF626 and Pediococcus acidilactici ZF627. The fermentation agent has high salt tolerance and high ester production ability. It is used for fermentation of fermented black beans, which can significantly improve the flavor, taste and aroma coordination of the fermented black beans, and improve the overall aroma, flavor and quality of the fermented black beans.

[0005] To achieve the above objectives, the inventors conducted in-depth research and completed the solution of the present invention after repeated research and demonstration, which is as follows:

[0006] In a first aspect, the present invention provides a composite lactic acid bacteria fermentation agent, wherein the composite fermentation agent comprises Staphylococcus carnosus ZF626 and Pediococcus acidilactici ZF627.

[0007] In a second aspect, the present invention provides a composite fermentation method, wherein the bacteria inoculated in the fermentation method include the above-mentioned Staphylococcus carnosus ZF626 and Pediococcus acidilactici ZF627.

[0008] In a third aspect, the present invention provides the use of the above-mentioned fermentation bacteria agent or composite fermentation method in fermented food processing.

[0009] Furthermore, the fermented food is any one or more types of fermented bean foods, and preferably the fermented bean food is fermented black beans.

[0010] In a fourth aspect, the present invention provides a method for preparing fermented black beans, wherein the method comprises adding the above-mentioned fermentation agent during the fermentation process of the fermented black beans for fermentation.

[0011] Furthermore, the fermentation agent is added in the early stage of fermentation.

[0012] Furthermore, the addition amount of the fermentation agent is 10 5 ~10 7 CFU / mL.

[0013] Furthermore, the ratio of Staphylococcus carnosus ZF626 to Pediococcus acidilactici ZF627 in the fermentation bacteria is 1:1.

[0014] In a fifth aspect, the present invention provides a fermented black beans, which is prepared by the preparation method described in the present invention.

[0015] One or more technical solutions provided in the embodiments of the present application have at least the following technical effects or advantages:

[0016] 1. The composite fermentation agent provided by the present invention has high salt tolerance and high ester production capacity, and has a strong ability to produce flavor compounds; it can be used in the production of high-salt fermented foods to enhance product flavor, thereby improving food quality.

[0017] 2. The composite fermentation agent provided by the present invention can significantly increase the content of total volatile flavor substances in fermented black beans by 71%, among which the relative content of key aroma substances such as alcohols, esters, phenols, and acids can be increased by 58.4%-132.3%.

[0018] 3. The composite fermentation agent provided by the present invention is used for fermenting black beans. The concentrations of seven key flavor substances in the black beans, such as isopentanol, ethyl acetate, γ-caprolactone, and guaiacol, are significantly increased. It is particularly noteworthy that γ-caprolactone, which was not detected in the control, was detected in the composite fermented black beans, and the content of guaiacol was increased by more than 27 times. In addition, the content of substances such as isopentanol and ethyl acetate was also increased by 48%-140%. The increase in the content of these flavor compounds can significantly improve the flavor, taste and aroma coordination of the black beans.

[0019] 4. The present invention provides a composite fermentation method for fermented black beans, which has a simple process and can be used for large-scale production. The overall flavor, taste and aroma coordination of the fermented black beans are significantly better than those of naturally fermented black beans and commercially available black beans, which not only improves the overall aroma and flavor of the black beans, but also makes the flavor and taste more rich, complex, layered and recognizable, significantly improving the overall flavor and quality of the black beans. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is the colony morphology of Staphylococcus carnosus ZF626;

[0021] Figure 2 This is the colony morphology of Pediococcus acidilactici ZF626;

[0022] The Staphylococcus carnosus ZF626 provided by the present invention has been deposited in the Guangdong Microbiological Culture Collection Center on July 17, 2024, with the address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City, Guangdong Province, and the collection number is GDMCC NO: 64878; the strain was received and registered by the collection center on July 17, 2024, and was detected as a viable strain by the collection center on July 17, 2024.

[0023] The Pediococcus acidilactici ZF627 provided by the present invention has been deposited in the Guangdong Microbiological Culture Collection Center on July 17, 2024, with an address of 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City, Guangdong Province, and a collection number of GDMCC NO: 64879; the strain was received and registered by the collection center on July 17, 2024, and was detected as a viable strain by the collection center on July 17, 2024. DETAILED DESCRIPTION

[0024] In order to facilitate the understanding of the present invention, the present invention will be described more fully below. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0025] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0026] Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional food-grade reagents, methods and equipment in the art.

[0027] The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more of the associated listed items.

[0028] The headspace solid phase microextraction-gas phase mass spectrometry (HS-SPME-GC-MS) technique used in the specification of the present invention is used to determine the volatile flavor substances in fermented black beans. The specific method is as follows:

[0029] (1) Sample processing

[0030] Weigh 2 g of the ground sample, place it in a 15 mL headspace bottle, add 3 g of sodium chloride and an appropriate amount of ultrapure water, then add 15 μL of 2-octanol (concentration 9.7 ppm) internal standard solution and mix evenly, place it in a 60 °C constant temperature incubator for oscillation for 30 min, insert a DVB / CAR / PDMS solid phase microextraction head for extraction for 30 min, and resolve at 250 °C for 1 min.

[0031] (2) GC-MS conditions

[0032] GC conditions: HP-INNOWax capillary column (60m×250μm); splitless injection mode, temperature program: 55°C for 5min, then increase to 230°C at 5°C / min and hold for 15min; carrier gas: helium at a flow rate of 1.2mL / min.

[0033] MS conditions: EI ion source; ion source stability of 250°C, quadrupole and mass spectrometer interface temperatures of 150°C and 280°C, respectively; electron energy 70 eV, full scan mode; mass scan range 29-500 m / z. The mass spectrum data of unknown volatile substances were compared and confirmed with the standard spectral library map NIST 2017, and peak identification was performed with similarity (SI) > 80 (the highest value is 100) as the standard.

[0034] (3) Quantitative analysis

[0035] The internal standard method was used to calculate the relative content of each component. The calculation formula is as follows:

[0036]

[0037] Wherein, C represents the relative content of a single component, mg / kg; A1 represents the peak area of ​​a single component; A2 represents the peak area of ​​the internal standard; C2 represents the internal standard mass concentration, μg / mL; V2 represents the internal standard volume, μL; and M represents the sample mass, g.

[0038] Example 1 Screening of target strains

[0039] 1. Processing of raw materials

[0040] Take a certain amount of flavored fermented black beans, crush them with a sterile mortar, weigh 10g of the crushed sample and add it to a 250mL conical flask containing 90mL sterile saline, and use a constant temperature shaking incubator to shake and culture at 30℃ and 200rpm for half an hour to allow the microorganisms in the sample to be fully suspended in the saline; draw the suspension and add sterile saline to dilute it step by step by 10 times to 10 -2 -10 -6 , make bacterial dilution liquid; select 2-3 bacterial suspensions with appropriate gradients, respectively draw 200 μL of liquid and add them to LB plates and MRS plates containing 20 mg / L natamycin, evenly spread them with a sterile spreading rod, and then invert and culture them in a 37°C incubator for 1-2 days; after clearly visible colonies grow on the LB plates and MRS plates, use a sterile inoculation loop to pick up single colonies with inconsistent morphology, inoculate them into corresponding LB or MRS culture media for streak culture at 37°C, and culture them in an incubator for 1-2 days; repeat this 2-3 times until the morphology of the colonies grown on the plates is basically consistent, and a pure strain of microorganisms is obtained.

[0041] Through the above-mentioned microbial separation and purification scheme, 12 purified strains were obtained from the flavored fermented black beans and conventionally preserved, and were numbered DC101-DC112 respectively.

[0042] 2. Preliminary screening of bacterial strains

[0043] The salt content during fermentation of fermented black beans is between 12-15%. In order to ensure that the target microorganism can maintain a good growth state during fermentation of fermented black beans, it is necessary to test its salt tolerance. The 12 purified strains isolated were inoculated into the corresponding LB or MRS culture medium for activation, and then a single colony was picked and inoculated into the LB or MRS liquid culture medium to culture until the middle of logarithmic growth; the seed liquid was inoculated into LB liquid culture medium and MRS liquid culture medium with salt contents of 8%, 12%, 16%, and 20% at a 2% inoculation rate, and cultured at 37°C and 200rpm for 3 days. After the fermentation was completed, the absorbance value OD at a wavelength of 600nm was measured. 600 , 3 parallels were set for each group. The test results are shown in Table 1:

[0044] Table 1 Growth of 12 purified strains in different salt media

[0045]

[0046]

[0047] As shown in Table 1, with the increase of salt content, the growth rate of all strains decreased, but strain DC103, strain DC104, strain DC105, strain DC107 and strain DC111 showed strong salt tolerance and could maintain relatively high biomass at a salt content of 12%-20%, so these 5 strains can be applied to the fermentation process of fermented black beans. These 5 strains were selected for rescreening.

[0048] 3. Lactic acid bacteria re-screening

[0049] The five strains initially screened were reactivated and cultured, inoculated into LB or MRS liquid culture medium, and cultured at 37°C for 24 h. The cells were collected by centrifugation, washed with sterile saline, and diluted to obtain a concentration of 10 9 -10 10 CFU / mL of bacterial suspension, and then inoculated into the rescreening medium (100mL of sterile fermented black bean extract, 0.5% acetic acid and 2% ethanol) at a 2% inoculation amount, and cultured at 37°C for 3 days, and the uninoculated rescreening medium was used as the control group. After the fermentation was completed, the content of ethyl acetate was detected by headspace solid phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS). The results are shown in Table 2:

[0050] Table 2 Results of rescreening of 5 highly salt-tolerant strains

[0051] Group Ethyl acetate (μg / L) Control group 13.68±0.54 DC103 fermentation group 60.15±3.11 DC104 fermentation group 70.78±4.21 DC105 fermentation group 201.52±4.68 DC107 fermentation group 50.73±2.32 DC111 fermentation group 174.67±6.17

[0052] As shown in Table 2, the ethyl acetate content in all fermentation groups was higher than that in the control group, indicating that these strains were able to synthesize ester compounds to a certain extent. Among them, the ethyl acetate production of strains DC105 and DC111 in the rescreening medium was significantly higher than that of other strains, showing a strong ester production ability.

[0053] The DC105 and DC111 strains obtained by the above screening were genetically identified as Pediococcus acidilactici and Staphylococcus carnosus, respectively, which can be used in the food fermentation industry. They were named Pediococcus acidilactici ZF627 and Staphylococcus carnosus ZF626, respectively, and were deposited in Guangdong Provincial Microbiological Culture Collection on July 17, 2024, with the deposit numbers GDMCC NO: 64879 and GDMCC NO: 64878, respectively.

[0054] Example 2 Test of fermented black beans by Pediococcus acidilactici ZF627 and Staphylococcus carnosus ZF626

[0055] (1) Soaking: Soak the screened soybeans at room temperature for 2-3 hours until the beans swell without wrinkles, feel firm, the bean skin is not easy to fall off, and there is no large amount of foam on the liquid surface;

[0056] (2) Steaming: Steam the soaked soybeans at 115°C for 20 min in a high-temperature and high-pressure autoclave;

[0057] (3) Koji making: After the soybeans are naturally cooled to 35-40°C, soy sauce koji and wheat flour are evenly sprinkled on the soybeans and stirred evenly. The ratio of koji to soybean dry weight is 0.1%, and the amount of wheat flour added is 100 times that of koji. The koji making temperature is controlled at 32-35°C, the koji making time is 48 hours, and the koji is turned every 12 hours;

[0058] (4) Stack fermentation: 12.5% ​​of the brine was mixed evenly with the koji material in a ratio of brine to koji material = 8:33, and fermented at 30°C for 30 days, with irregular stirring during the fermentation process;

[0059] (5) Inoculation: Staphylococcus carnosus ZF626 and Pediococcus acidilactici ZF627 were cultured to the middle and late logarithmic growth stages, the bacterial precipitates were collected by centrifugation, washed 2-3 times with physiological saline, and the bacterial precipitates were resuspended to a cell concentration of 5×10 7 CFU / mL and then mixed at a ratio of 1:1. When the fermentation time of fermented black beans reaches 20h, the mixed bacterial solution is inoculated into the fermented black beans at an inoculation rate of 2% for fermentation; no inoculation and single inoculation of Staphylococcus carnosus ZF626 and Pediococcus acidilactici ZF627 are used as controls;

[0060] (6) Detection and analysis: After the fermentation was completed, samples were collected to determine the physical and chemical indicators and volatile flavor substance content of the fermented fermented black beans, and the results were recorded in Tables 3-4; and based on the orthogonal partial least squares discriminant analysis (OPLS-DA), volatile compounds with variable weight values ​​(VIP) less than 1 and Student's t-test P values ​​less than 0.05 were screened to obtain some key differential aroma compounds, and their concentration measurement results are recorded in Table 5.

[0061] (7) Sensory evaluation: The above fermented black beans were used for flavor evaluation with a commercially available fermented black beans. 30 trained professional sensory evaluators were invited to conduct sensory evaluation on the five dimensions of flavor, color, taste, appearance and aroma harmony of different fermented black beans. The samples were scored and the average value was taken. The sample scores and final sensory evaluation results are recorded in Table 6.

[0062] Table 3 Results of physical and chemical index determination of fermented black beans by different strains

[0063]

[0064]

[0065] As shown in Table 3, the reducing sugar content of the ZF626 and ZF627 composite fermentation group was significantly higher than that of other groups, reaching 10.37 g / 100 g, which was more than 80% higher than that of the CK group. This shows that the mixed inoculation of ZF626 and ZF627 in the early stage of fermentation of fermented black beans can not only accelerate the process of converting polysaccharides such as starch into monosaccharides, but also help to generate more reducing sugar substances, which may have a positive effect on improving the sweetness of fermented black beans and enhancing the mellow flavor through the Maillard reaction. Overall, appropriate adjustment of the microbial combination can effectively improve the key flavor components of fermented black bean products.

[0066] Table 4 Detection of volatile flavor substances in fermented black bean curd samples

[0067]

[0068] As shown in Figure 4, the content of total volatile flavor substances in fermented black beans inoculated with ZF626 and ZF627 was significantly higher than that in the control group, and the increase rate was 71% compared with natural fermentation, among which the relative content increase rates of key aroma substances such as alcohols, esters, phenols, and acids reached 58.44%, 132.30%, 73.45%, and 72.48%, respectively. Alcohol compounds are one of the important aroma components in black beans, which can give the product a rich mellow aroma; ester compounds make black beans have a richer fruity and floral aroma; phenolic compounds are usually associated with woody, smoky, and spicy aromas in food. High phenolic compound content may give black beans a more complex woody or smoky aroma, enhancing the depth and layering of its flavor; acid compounds help to enhance the umami and acidity of black beans, making its flavor more distinct.

[0069] Table 5 Different aroma substances in fermented fermented black bean samples

[0070]

[0071] As shown in Table 5, the composite fermentation of fermented black beans by inoculation of ZF626 and ZF627 can significantly increase the content of seven key flavor substances in the fermented black beans, including isopentanol, ethyl acetate, γ-caprolactone, and guaiacol, and can significantly improve the overall aroma and flavor of the fermented black beans.

[0072] First of all, γ-caprolactone is a special flavor compound detected in compound fermented black beans, with a content of 1.31 mg / kg, which was not detected in the control group or the content was extremely low; it has herbal aroma, sweet aroma and caramel aroma, which can add a unique aroma to black beans, enrich its flavor levels, cover up unpleasant odors, and at the same time make the taste of black beans more mellow and round.

[0073] Secondly, the content of guaiacol in the composite fermented black beans reached 0.86 mg / kg, which was 28.7 times that of the control group. Guaiacol is an important phenolic compound. High guaiacol content can give black beans a more complex woody or smoky aroma, making the aroma of black beans more complex, layered and recognizable. At the same time, it can cooperate with volatile substances such as alcohols, aldehydes, esters, etc. to enhance or adjust the aroma of black beans, making the aroma of black beans more intense and coordinated.

[0074] Again, the content of isopentanol in ZF626 and ZF627 compound fermented douchi reached 0.79 mg / kg, which is 2.14 times that of the CK group. Isopentanol is a fruity compound that can significantly enhance the fruity characteristics of douchi. The content of ethyl acetate in the ZF626 and ZF627 compound fermented group was 0.12 mg / kg, which is 2.4 times that of the CK group. Ethyl acetate has a fruity aroma and can increase the sweetness and fruity aroma of douchi, making the taste of douchi richer and fuller.

[0075] Finally, the contents of 1-octen-3-ol, 3-methylbutanal, and 2-methylbutanal in the fermented black beans inoculated with ZF626 and ZF627 were also significantly increased, which was 1.4-1.5 times that of the CK group; among them, 1-octen-3-ol has a mushroom aroma, which can increase the aroma level and recognition of black beans, 3-methylbutanal has a fruity aroma, and 2-methylbutanal has an almond smell, which can increase the aroma intensity and complexity of black beans.

[0076] Table 6 Sensory evaluation results of different fermented black beans

[0077] Group Flavor Color Taste Appearance Aroma coordination Comprehensive evaluation Commercially available control 6.1 5.2 6.8 6.5 5.7 6.5 Group 1 5.8 4.9 6.6 6.2 5.4 6.3 Group 2 5.5 6.2 6.5 6.1 6.1 6.4 Group 3 7.1 6.3 6.5 6.4 6.5 6.9 Group 4 7.5 6.1 7.6 6.9 7.8 7.8

[0078] Note: Each indicator is scored on a 9-point scale, with the larger the value, the more prominent the indicator.

[0079] The results of the sensory evaluation experiment show that the complex fermented black beans inoculated with ZF626 and ZF627 performed best in multiple sensory indicators, especially in flavor, taste and aroma coordination, which were significantly better than other groups. This is consistent with the results of volatile compound analysis, indicating that inoculating ZF626 and ZF627 in the early stage of black bean fermentation can significantly improve the flavor and overall quality of black bean.

[0080] In summary, by mixed inoculation of Staphylococcus carnosus ZF626 and Pediococcus acidilactici ZF627 in the early stage of fermentation of fermented black beans, the contents of various key volatile compounds in fermented black beans were significantly increased, including the total content of flavor substances such as alcohols, esters, phenols and acids, especially the contents of isopentanol, 1-octen-3-ol, ethyl acetate, γ-caprolactone, 3-methylbutanal, 2-methylbutanal and guaiacol were significantly increased. The increase of these compounds can significantly improve the flavor, taste and aroma coordination of fermented black beans, which not only improves the overall aroma and flavor of fermented black beans, but also makes its flavor and taste more rich, complex, layered and recognizable, significantly improving the overall flavor and quality of fermented black beans.

[0081] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A composite fermentation agent, characterized in that: The fermentation bacteria agent comprises Staphylococcus carnosus ZF626 with a preservation number of GDMCC NO: 64878 and Pediococcus acidilactici ZF627 with a preservation number of GDMCC NO: 64879.

2. A composite fermentation method, characterized in that: The bacteria inoculated in the fermentation method include Staphylococcus carnosus ZF626 and Pediococcus acidilactici ZF627 according to claim 1.

3. Use of the fermentation agent according to claim 1 or the composite fermentation method according to claim 2 in fermented food processing.

4. The use according to claim 3, characterized in that The fermented food is any one or more types of fermented bean foods.

5. The use according to claim 4, characterized in that The fermented bean food is fermented black beans.

6. A method for preparing fermented black beans, characterized in that: The method is a method of adding the fermentation agent according to claim 1 during the fermentation process of fermented black beans to carry out fermentation.

7. The preparation method according to claim 6, characterized in that: The fermentation agent is added in the early stage of fermentation.

8. The preparation method according to claim 6, characterized in that: The addition amount of the fermentation agent is 10 5 ~10 7 CFU / mL.

9. The preparation method according to claim 6, characterized in that: The ratio of Staphylococcus carnosus ZF626 to Pediococcus acidilactici ZF627 in the fermentation bacteria is 1:

1.

10. A fermented black bean, characterized in that: The fermented black beans are prepared by the preparation method described in any one of claims 6-9.

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