Soy cheese starter with deodorization and flavor enhancement function and application thereof

Abstract

The present application relates to a kind of soy cheese ferment with the function characteristics of removing fishy smell and enhancing flavor and its application.The soy cheese ferment of the present application includes a kind of ferment Pichia RS-13 (preservation number: CCTCC NO: M 2020964) and lactococcus FR23 (preservation number: CCTCC NO: M 20242058);The present application utilizes the complement of different strains in weakening bean fishy smell and enhancing milk aroma, fruit aroma flavor intensity and other aroma-producing performance, to make the type, content and sensory evaluation result of key aroma component in fermented soy cheese as main index, utilize the strain combination after compounding can achieve the purpose of removing fishy smell and enhancing flavor complementary advantage, flavor optimization.The soy cheese ferment of the present application is prepared, and soy cheese milk aroma, fruit aroma are prominent, and bean fishy smell is reduced, and sensory performance is good and stable, and thus the flavor of fermented soy cheese product produced is better, and the overall sensory acceptance and flavor quality of fermented soy cheese are improved.

Description

Technical Field

[0001] This invention belongs to the field of food fermentation technology, and in particular relates to a soybean cheese fermentation agent with deodorizing and aroma-enhancing properties and its application. Background Technology

[0002] In recent years, driven by health, sustainability, and animal welfare concerns, consumer interest in plant-based cheese has grown significantly. Soybeans, with their wide availability and high protein content, are an excellent raw material for plant-based cheese development. However, unpleasant beany odors often develop in soybean products during processing, directly impacting the edible value of plant-based cheese and thus limiting its market prospects. The beany odor in soybean foods is primarily due to the activation of lipoxygenases in soybeans by oxygen and water during the soybean grinding process. This oxidizes polyunsaturated fatty acids (mainly linoleic acid and linolenic acid) into hydroperoxides, which further degrade into various low-molecular-weight alcohols, aldehydes, ketones, acids, and amines—volatile components. These small-molecule compounds often possess varying degrees of off-odor, resulting in the beany smell, such as hexanal, benzaldehyde, 2-pentylfuran, and 1-octen-3-ol.

[0003] Studies have shown that microbial fermentation can reduce the beany odor of soybean products. This is mainly achieved by relying on the aroma-producing and enzymatic hydrolysis capabilities of specific microorganisms to generate flavorful metabolites (such as 3-methylbutyraldehyde, 2-nonanone, and hexyl acetate) to mask the beany odor, or to degrade and transform beany odor substances, thereby reducing the beany odor and improving the overall acceptability of the sample.

[0004] Chinese patent CN113151018B discloses a fermented Pichia pastoris RS-13 and its applications, with accession number CCTCCNO: M 2020964 and Latin name Pichia fermentans RS-13, which was deposited at the China Center for Type Culture Collection on December 24, 2020. Experiments have shown that this fermented Pichia pastoris can reduce the beany odor in soybean residue to a level imperceptible to the human body, and reduce the content of beany odor compounds to 1 / 40 of that in unfermented soybean residue. Using fermented Pichia pastoris RS-13 in soybean residue beverage products can produce beany residue beverages without a beany odor. Therefore, it can effectively solve the technical problems of low utilization rate and limited product development caused by the strong beany odor of soybean residue, and has good application prospects.

[0005] Chinese patent CN116982659A discloses a low-beany-flavor pea protein plant-based cheese and its preparation method. The pea protein plant-based cheese provided is prepared by dry-heat mixing pea protein powder and potato starch in a specific ratio to obtain a mixed powder with a reduced beany flavor. This mixed powder is then added to a proportionally prepared oil-water mixture, along with thickeners, emulsifiers, and other auxiliary ingredients, and heated and stirred until the mixture becomes a smooth, viscous liquid. Pichia pastoris is then introduced for fermentation, stopping when the pH reaches 5.0-5.2. The mixture is then poured into molds and refrigerated to obtain a pea protein plant-based cheese with a further reduced beany flavor. This method simultaneously employs dry-heat mixing and solid-state fermentation techniques to remove the beany flavor from the pea protein plant-based cheese. Compared to ordinary pea protein plant-based cheese, the beany flavor is significantly reduced, and the fermented flavor is enhanced.

[0006] However, both of the aforementioned patents utilize Pichia fermentans RS-13 alone to reduce the beany taste.

[0007] Currently, most dairy starter cultures have been optimized for milk substrates, but they are not suitable for non-dairy substrates. The strains used for fermentation in soybean products that can reduce the beany taste are mostly yeasts, lactic acid bacteria, and molds. However, each of these strains has some shortcomings in single-strain fermentation. There is little research on the use of compound strains for fermentation to reduce the beany taste of soybean products, and the proportion of compound strains added also needs further exploration. Summary of the Invention

[0008] The technical problem to be solved by this invention is that soybean cheese has a strong beany smell and lacks flavor compared with traditional cheese. Existing cheese starter is not suitable for plant-based cheese substrates, and single-strain fermentation results in insufficient flavor.

[0009] To address the aforementioned technical problems, this invention provides a soybean cheese fermentation agent with deodorizing and aroma-enhancing properties, and its application.

[0010] The objective of this invention is achieved through the following technical solution:

[0011] This invention provides a soybean cheese starter with deodorizing and aroma-enhancing properties, which is a bacterial suspension containing Pichia pastoris RS-13 and Pediococcus lactis FR23;

[0012] The Pichia fermentans RS-13 has the accession number CCTCC NO: M2020964, is deposited at the China Center for Type Culture Collection on December 24, 2020, and is located at Wuhan University, Wuhan, China.

[0013] The accession number of Pediococcus acidilactici FR23 is CCTCC NO: M20242058. The depositary institution is the China Center for Type Culture Collection, the deposit date is September 24, 2024, and the deposit address is Wuhan University, Wuhan, China.

[0014] In one embodiment of the present invention, the soybean cheese starter contains a total viable count of at least 1 × 10⁻⁶ Pichia pastoris RS-13 and Pediococcus lactis FR23. 9 CFU / mL.

[0015] In one embodiment of the present invention, the soybean cheese starter is composed of a mixture of Pichia pastoris RS-13 bacterial suspension and Pediococcus lactis FR23 bacterial suspension, wherein the volume ratio of the Pichia pastoris RS-13 bacterial suspension to the Pediococcus lactis FR23 bacterial suspension is 1:1 to 100:1.

[0016] Preferably, the volume ratio of the Pichia pastoris RS-13 suspension to the Pediococcus lactis FR23 suspension is 50:1.

[0017] The present invention further provides the application of Pichia pastoris RS-13 or Pediococcus lactis FR23 in the preparation of soybean cheese starter, and the prepared soybean cheese starter has the function of removing fishy smell and enhancing aroma.

[0018] The present invention further provides a method for preparing the soybean cheese starter with deodorizing and aroma-enhancing properties, comprising the following steps:

[0019] Step 1: Streak the Pichia pastoris RS-13 culture solution on YPD solid medium plates and streak the Pediococcus lactis FR23 culture solution on MRS solid medium plates until single colonies grow.

[0020] Step 2: Inoculate the single colony of Pichia pastoris RS-13 obtained in Step 1 into liquid culture medium containing YPD broth and carry out liquid culture. Inoculate the single colony of Pediococcus lactis FR23 obtained in Step 1 into liquid culture medium containing Pediococcus lactis FR23 broth and carry out liquid culture. The culture media of Pichia pastoris RS-13 and Pediococcus lactis FR23 are obtained respectively.

[0021] Step 3: Centrifuge the culture medium of Pichia pastoris RS-13 and the culture medium of Pediococcus lactis FR23 separately, remove the supernatant, wash the cells with sterile water, add an equal volume of sterile water to the centrifuge tube, vortex to obtain the bacterial suspensions of Pichia pastoris RS-13 and Pediococcus lactis FR23 respectively.

[0022] Step 4: Mix the bacterial suspension of Pichia pastoris RS-13 obtained in Step 3 with the bacterial suspension of Pietrocarpus lactis FR23 to obtain a soybean cheese starter with deodorizing and aroma-enhancing properties.

[0023] In a specific embodiment of the present invention, the preparation method of the soybean cheese fermentation agent with deodorizing and aroma-enhancing properties specifically includes the following steps:

[0024] Step 1: Take out the preserved Pichia pastoris RS-13 and Pediococcus lactis FR23 bacterial suspensions from the glycerol preservation tubes respectively. Use an inoculation loop to take the Pichia pastoris RS-13 bacterial suspension and streak it on a YPD solid medium plate. Use an inoculation loop to take the Pediococcus lactis FR23 bacterial suspension and streak it on an MRS solid medium plate. Then, incubate them separately in an incubator at a constant temperature until single colonies grow.

[0025] Step 2: Use an inoculation loop to pick up one loopful of single colonies of the two strains obtained in Step 1, and then inoculate them into conical flasks containing YPD and MRS broth liquid medium, respectively. Place them in a constant temperature incubator for culture to obtain culture media of Pichia pastoris RS-13 and Pietrocarpus FR23, respectively.

[0026] Step 3: Centrifuge the culture media of the two strains of Pichia pastoris RS-13 and Pietrocarpus lactis FR23 separately, remove the supernatant, wash the cells with sterile water, add an equal volume of sterile water to the centrifuge tube, and vortex to obtain the bacterial suspensions of the two strains.

[0027] Step 4: Mix the two bacterial suspensions obtained in Step 3 in a certain proportion to obtain a soybean cheese fermentation agent with deodorizing and aroma-enhancing functions.

[0028] In one embodiment of the present invention, the YPD solid culture medium comprises: 20.0 g / L peptone, 20.0 g / L glucose, 10.0 g / L yeast extract and 13.5 g / L bacterial agar powder.

[0029] In one embodiment of the present invention, the MRS solid culture medium comprises: 10.0 g / L peptone, 10.0 g / L beef meal, 5.0 g / L yeast extract, 20.0 g / L glucose, 0.1 g / L magnesium sulfate, 5.0 g / L sodium acetate, 2.0 g / L ammonium citrate, 2.0 g / L dipotassium hydrogen phosphate, 0.05 g / L manganese sulfate, 1.0 g / L Tween 80, and 13.5 g / L bacterial agar powder.

[0030] In one embodiment of the present invention, in step 1, the streak culturing conditions are: temperature 37°C, time 24-48h.

[0031] In one embodiment of the present invention, in step 2, the YPD liquid culture medium consists of: 20.0 g / L peptone, 20.0 g / L glucose, and 10.0 g / L yeast extract.

[0032] In one embodiment of the present invention, in step 2, the composition of the MRS liquid culture medium is as follows: peptone 10.0 g / L, beef meal 10.0 g / L, yeast powder 5.0 g / L, glucose 20.0 g / L, magnesium sulfate 0.1 g / L, sodium acetate 5.0 g / L, ammonium citrate 2.0 g / L, dipotassium hydrogen phosphate 2.0 g / L, manganese sulfate 0.05 g / L, and Tween 80 1.0 g / L.

[0033] In one embodiment of the present invention, in step 2, the conditions for liquid culture are: temperature 37°C, time 10-15h.

[0034] In one embodiment of the present invention, in step 3, the centrifugation conditions are: temperature 4°C, rotation speed 10000 rpm, centrifugation time 5 min, and washing with sterile water 2-3 times.

[0035] In one embodiment of the present invention, in step 3, the condition for vortex oscillation is: vortex oscillation at 3000-6000 rpm for 5 minutes.

[0036] The present invention further provides the application of the soybean cheese starter with deodorizing and aroma-enhancing properties in the preparation of fermented soybean cheese.

[0037] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0038] This invention provides a soybean cheese starter with deodorizing and aroma-enhancing functions, composed of Pichia pastoris RS-13 and Pediococcus lactis FR23. Research has shown that Pediococcus lactis FR23 has deodorizing and aroma-enhancing effects. Therefore, the combination of Pichia pastoris RS-13 and Pediococcus lactis FR23 results in a lower content of beany-smelling substances, and their different aroma-enhancing effects lead to a richer aroma. Soybean cheese fermented using this combination of bacteria exhibits lower levels of beany-smelling substances such as hexanal, benzaldehyde, 2-pentylfuran, and 1-octen-3-ol, while containing higher levels of milky and fruity aroma substances such as 3-methylbutyraldehyde, 2-nonanone, and hexyl acetate.

[0039] This invention utilizes the complementary properties of different strains in reducing beany odor and enhancing the aroma of milk and fruit. The main indicators are the types, contents, and sensory evaluation results of key aroma components in the fermented soybean cheese. By using the compounded strain combination, the complementary advantages of deodorization and aroma enhancement can be achieved, and the flavor can be optimized.

[0040] The soybean cheese fermentation agent of this invention produces soybean cheese with prominent milky and fruity aromas, reduced beany odor, and good and stable sensory properties. The resulting fermented soybean cheese products have superior flavor, enhancing the overall sensory appeal and flavor quality. This application solves the flavor problem in the development of plant-based foods through endogenous flavor enhancement, while also meeting current consumer demand for green and healthy foods, aligning with the trend of "clean label" products. Attached Figure Description

[0041] Figure 1 This is a flowchart of the soybean cheese fermentation process.

[0042] Figure 2(a) shows the sensory evaluation acceptance radar chart of different soy cheeses;

[0043] Figure 2(b) is a radar chart showing the sensory evaluation results of different soybean cheeses.

[0044] Figure 3 A bar chart showing the accumulation of volatile flavor compounds in soybean cheese fermented with different proportions of different microbial strains.

[0045] Figure 4 Heat map of volatile flavor compounds in soybean cheese fermented with different proportions of different microbial strains;

[0046] Figure 5 The changes in aroma substance content in soybean cheese fermented with different strains of bacteria.

[0047] Preservation Instructions

[0048] The Pichia fermentans RS-13 has the accession number CCTCC NO: M2020964, is deposited at the China Center for Type Culture Collection on December 24, 2020, and is located at Wuhan University, Wuhan, China.

[0049] The accession number of Pediococcus acidilactici FR23 is CCTCC NO: M20242058. The depositary institution is the China Center for Type Culture Collection, the deposit date is September 24, 2024, and the deposit address is Wuhan University, Wuhan, China. Detailed Implementation

[0050] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments.

[0051] The YPD solid culture medium used in the various embodiments of the present invention consists of: 20.0 g / L peptone, 20.0 g / L glucose, 10.0 g / L yeast extract and 13.5 g / L bacterial agar powder.

[0052] The MRS solid culture medium used in the various embodiments of the present invention comprises: 10.0 g / L peptone, 10.0 g / L beef meal, 5.0 g / L yeast extract, 20.0 g / L glucose, 0.1 g / L magnesium sulfate, 5.0 g / L sodium acetate, 2.0 g / L ammonium citrate, 2.0 g / L dipotassium hydrogen phosphate, 0.05 g / L manganese sulfate, 1.0 g / L Tween 80, and 13.5 g / L bacterial agar powder.

[0053] The YPD liquid culture medium used in the various embodiments of the present invention consists of: 20.0 g / L peptone, 20.0 g / L glucose, and 10.0 g / L yeast extract.

[0054] The MRS liquid culture medium used in the various embodiments of the present invention comprises: 10.0 g / L peptone, 10.0 g / L beef meal, 5.0 g / L yeast extract, 20.0 g / L glucose, 0.1 g / L magnesium sulfate, 5.0 g / L sodium acetate, 2.0 g / L ammonium citrate, 2.0 g / L dipotassium hydrogen phosphate, 0.05 g / L manganese sulfate, and 1.0 g / L Tween 80.

[0055] The aroma compounds in the fermented soybean cheese obtained in the various embodiments of the present invention were determined by gas chromatography-mass spectrometry (GC-MS), and the steps are as follows:

[0056] Weigh 5g of fermented soybean cheese sample into a 15mL extraction bottle, add 5μL (220mg / L) of 2-octanol internal standard, equilibrate in a 60℃ constant temperature water bath for 10min, and then insert the SPME extraction device, which has been aged for 20min, into the headspace bottle to extract the fermented soybean cheese sample for 40min.

[0057] After extraction, the extraction head was inserted into the gas chromatograph injection port at 250℃ for 5 minutes for analysis. The extracted volatile compounds were then analyzed and identified by GC-MS. The gas chromatographic conditions were as follows: column conditions: HP-innowax (60m×0.25mm×0.25μm); injection port temperature: 250℃; temperature program: initial temperature 40℃, hold for 3 min, increase to 120℃ at 5℃ / min, hold for 4 min, increase to 200℃ at 8℃ / min, hold for 8 min, increase to 240℃ at 10℃ / min, hold for 10 min; detector temperature: 250℃; carrier gas: helium, flow rate: 1 mL / min; injection method: splitless injection. Mass spectrometry conditions: EI ionization source, electron energy 70 eV, scan range 30-300 amu, ion source temperature 230℃; quadrupole temperature: 150℃; after each analysis, the extraction head needs to be aged in an aging instrument at 250℃ for 20 min, and each sample is analyzed 3 times.

[0058] Example 1

[0059] Pichia fermentans RS-13 was isolated from the traditional Chinese fermented food, milk fan, by the Food Flavor Regulation Innovation Team of the School of Flavor and Fragrance Technology and Engineering, Shanghai University of Applied Technology. It is deposited at the China Center for Type Culture Collection (CCTCCM2020964), Wuhan University, Wuhan, China, 430072, China. The isolation and screening of Pichia fermentans RS-13 is described in patent CN113151018A.

[0060] The isolation and screening of strain FR23 were carried out according to the following steps:

[0061] (1) Under sterile conditions, take 1g of sample (the sample is Guizhou fermented bean curd, which is naturally fermented locally and stored at 4℃ until sampling) into a sterile test tube, add 9ml of physiological saline to the sterile test tube, mix well to obtain a dilution of 10. -1 bacterial solution;

[0062] (2) Take the dilution obtained in step (1) as 10. -1 1 mL of bacterial culture was added to another sterile test tube, and 9 mL of physiological saline was added to the test tube. The mixture was then stirred to obtain a dilution gradient of 10. -2 The bacterial culture was prepared; the above steps were repeated 4 times to obtain 10... -3 10 -4 10 -5 and 10 -6 Four gradients of bacterial solutions;

[0063] (3) The 10 obtained in step (2) -3 ~10 -6 Take 0.1 mL of bacterial solution from each of the four gradients and spread it onto MRS solid medium plates. Incubate at 37°C with the plates inverted for 24-48 h.

[0064] (4) Use a sterile toothpick to pick up a single colony of the isolate obtained after plate culture in step (3), inoculate it into MRS liquid medium, and incubate it in a 37℃ incubator for 12h to obtain the fermentation broth of the isolate.

[0065] (5) Centrifuge the fermentation broth of the isolates corresponding to step (4) at 4°C at 10,000 rpm for 5 min. After centrifugation, discard the supernatant and add an equal volume of sterile water. Centrifuge at 10,000 rpm for 5 min and discard the supernatant. Repeat this washing process 2-3 times. Finally, add an equal volume of sterile water and vortex at 6,000 rpm for 5 min to prepare a bacterial suspension for later use.

[0066] (6) Take 10 parts by weight of soy protein isolate, 100 parts by weight of water, 10 parts by weight of glucose, and 5.56 parts by weight of white sugar. Stir at 95°C and 500 r / min for 10 min. Then fill the container and sterilize it by heating in a water bath at 85°C for 10 min. Then cool it to 37°C to obtain sterile soy cheese inoculation solution.

[0067] (7) Take a portion of the bacterial suspension from step (5) and another portion of the bacterial suspension prepared by Pichia fermentans RS-13. Inoculate the bacterial suspension into the soybean cheese inoculation solution in (6) at an inoculation amount of 6% (v / v) under sterile conditions. Ferment at 37°C for 8 hours and then refrigerate at 4°C to obtain fermented soybean cheese curd.

[0068] Sensory evaluation and GC-MS identification were performed on the two types of fermented soybean cheese mentioned above. The results showed that both strains had deodorizing effects. The fermented soybean cheese made from strain RS-13 had almost no beany odor and produced some isoamyl alcohol, phenylethyl alcohol, and other aromatic compounds, but its overall flavor was rather bland and its curdling was poor. The fermented soybean cheese made from strain FR23 had a richer aroma, with more pronounced milky and fruity flavors, and higher levels of 3-methylbutyraldehyde and 2,3-butanediol. While the beany odor was reduced compared to the unfermented cheese, it was still noticeable. Ultimately, these two strains with deodorizing and aroma-enhancing functions were selected for blending and fermenting soybean cheese.

[0069] Biological identification of strain FR23 was performed, including PCR amplification of genomic DNA and 16S rRNA sequence detection; the identification results of strain RS-13 are described in patent CN113151018A. The primers used for PCR amplification were 27F (5'-GAGAGTTTGATCCTGGCTCAG-3') (SEQ ID NO: 1) and 1492r (5'-TACGGCTACCTTGTTACGAC-3') (SEQ ID NO: 2). The amplification system consisted of 2 μL of 27F primer, 2 μL of 1492r primer, 2 μL of bacterial culture, 19 μL of ddH2O, and 25 μL of 2×Gold Star Best Mixed. The PCR reaction procedure was as follows: (1) 95℃ / 10min, (2) 94℃ / 30s, (3) 55℃ / 30s, (4) 72℃ / 1.5min, repeating steps (2) to (4) for 30 cycles, (5) 72℃ / 5min, (6) storage at 4℃. After PCR amplification, agarose gel electrophoresis was performed. The appearance of clear bands in the measured fragments on the gel imaging instrument indicated that the PCR was successful. The remaining bacterial samples from the PCR were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The sequencing results showed that strain FR23 was Pediococcus acidilactici. The strain was then deposited at the China Center for Type Culture Collection (CCTCC M 20242058).

[0070] Example 2

[0071] A soybean cheese starter with deodorizing and aroma-enhancing functions comprises a bacterial suspension prepared by Pichia fermentans RS-13 and Pediococcus acidilactici FR23, wherein the volume ratio of the bacterial suspension prepared by Pichia fermentans RS-13 to the bacterial suspension prepared by Pediococcus acidilactici FR23 is 1:1 to 100:1;

[0072] The preparation method of soybean cheese starter includes the following steps:

[0073] (1) Take out the Pichia fermentans RS-13 and Pediococcus acidilactici FR23 bacterial suspensions from the glycerol storage tubes respectively. Use an inoculation loop to take the Pichia fermentans RS-13 bacterial suspension and streak it on a YPD solid medium plate. Use an inoculation loop to take the Pediococcus acidilactici FR23 bacterial suspension and streak it on an MRS solid medium plate. Then place them in an incubator at 37℃ for 24h until single colonies grow.

[0074] (2) Pick one loop of single colonies of the two strains obtained in step 1 with an inoculation loop, and then inoculate them into conical flasks containing YPD and MRS broth liquid culture medium respectively. Incubate them in a 37℃ incubator for 24h to obtain the culture medium of the two strains respectively.

[0075] (3) Centrifuge the culture media of the two strains obtained in (2) at 4℃, 10000 rpm for 5 min. After centrifugation, discard the supernatant, add an equal volume of sterile water, centrifuge at 10000 rpm for 5 min, discard the supernatant, and repeat this washing process 2-3 times. Finally, add an equal volume of sterile water and vortex at 6000 rpm for 5 min to prepare a bacterial suspension for later use. The bacterial content in the bacterial suspension is 1×10⁻⁶. 9 CFU / mL;

[0076] (4) The bacterial suspensions of Pichia pastoris RS-13 and Pediococcus lactis FR23 obtained in (3) are mixed at a volume ratio of 1:1 to 100:1 to obtain a soybean cheese starter with deodorizing and aroma-enhancing functions. The total viable count in the starter is 1×10⁻⁶. 9 CFU / mL.

[0077] Example 3

[0078] The application of the soybean cheese starter with deodorizing and aroma-enhancing functions obtained in Example 2 in fermented soybean cheese specifically includes the following steps, which can be participated in. Figure 1 The soybean cheese fermentation process flow shown is as follows:

[0079] Take 10 parts by weight of soy protein isolate, 100 parts by weight of water, 10 parts by weight of glucose, and 5.56 parts by weight of white sugar. Stir at 95°C and 500 r / min for 10 min. Then fill the container and sterilize it by heating in a water bath at 85°C for 10 min. Then cool it to 37°C to obtain the soy cheese fermentation liquid.

[0080] The soybean cheese starter described in Example 2 was inoculated into the soybean cheese fermentation liquid under sterile conditions, with a total inoculation amount of 6% (v / v), and fermented at 37°C for 8 hours. After that, it was refrigerated at 4°C to obtain fermented soybean cheese curd.

[0081] Among them, the soybean cheese starter with deodorizing and aroma-enhancing functions contains a 100:1 volume ratio of Pichia pastoris RS-13 and Pietrocarpus FR23 bacterial suspension, with a bacterial content of 1×10⁻⁶ in the suspension. 9 CFU / mL.

[0082] Example 4

[0083] The application of the soybean cheese starter with deodorizing and aroma-enhancing functions obtained in Example 2 in fermented soybean cheese includes the following steps:

[0084] Take 10 parts by weight of soy protein isolate, 100 parts by weight of water, 10 parts by weight of glucose, and 5.56 parts by weight of white sugar. Stir at 95°C and 500 r / min for 10 min. Then fill the container and sterilize it by heating in a water bath at 85°C for 10 min. Then cool it to 37°C to obtain the soy cheese fermentation liquid.

[0085] The soybean cheese starter described in Example 2 was inoculated into the soybean cheese fermentation liquid under sterile conditions, with a total inoculation amount of 6% (v / v), and fermented at 37°C for 8 hours. After that, it was refrigerated at 4°C to obtain fermented soybean cheese curd.

[0086] Among them, the soybean cheese starter with deodorizing and aroma-enhancing functions has a volume ratio of Pichia pastoris RS-13 and Pietrocarpus FR23 bacterial suspensions of 50:1.

[0087] Example 5

[0088] The application of the soybean cheese starter with deodorizing and aroma-enhancing functions obtained in Example 2 in fermented soybean cheese includes the following steps:

[0089] Take 10 parts by weight of soy protein isolate, 100 parts by weight of water, 10 parts by weight of glucose, and 5.56 parts by weight of white sugar. Stir at 95°C and 500 r / min for 10 min. Then fill the container and sterilize it by heating in a water bath at 85°C for 10 min. Then cool it to 37°C to obtain the soy cheese fermentation liquid.

[0090] The soybean cheese starter described in Example 2 was inoculated into the soybean cheese fermentation liquid under sterile conditions, with a total inoculation amount of 6% (v / v), and fermented at 37°C for 8 hours. After that, it was refrigerated at 4°C to obtain fermented soybean cheese curd.

[0091] Among them, the soybean cheese starter with deodorizing and aroma-enhancing functions has a volume ratio of Pichia pastoris RS-13 and Pietrococcus FR23 bacterial suspensions of 10:1.

[0092] Example 6

[0093] The application of the soybean cheese starter with deodorizing and aroma-enhancing functions obtained in Example 2 in fermented soybean cheese includes the following steps:

[0094] Take 10 parts by weight of soy protein isolate, 100 parts by weight of water, 10 parts by weight of glucose, and 5.56 parts by weight of white sugar. Stir at 95°C and 500 r / min for 10 min. Then fill the container and sterilize it by heating in a water bath at 85°C for 10 min. Then cool it to 37°C to obtain the soy cheese fermentation liquid.

[0095] The soybean cheese starter described in Example 2 was inoculated into the soybean cheese fermentation liquid under sterile conditions, with a total inoculation amount of 6% (v / v), and fermented at 37°C for 8 hours. After that, it was refrigerated at 4°C to obtain fermented soybean cheese curd.

[0096] Among them, the soybean cheese starter with deodorizing and aroma-enhancing functions has a volume ratio of Pichia pastoris RS-13 and Pietrocarpus FR23 bacterial suspensions of 5:1.

[0097] Example 7

[0098] The application of the soybean cheese starter with deodorizing and aroma-enhancing functions obtained in Example 2 in fermented soybean cheese includes the following steps:

[0099] Take 10 parts by weight of soy protein isolate, 100 parts by weight of water, 10 parts by weight of glucose, and 5.56 parts by weight of white sugar. Stir at 95°C and 500 r / min for 10 min. Then fill the container and sterilize it by heating in a water bath at 85°C for 10 min. Then cool it to 37°C to obtain the soy cheese fermentation liquid.

[0100] The soybean cheese starter described in Example 2 was inoculated into the soybean cheese fermentation liquid under sterile conditions, with a total inoculation amount of 6% (v / v), and fermented at 37°C for 8 hours. After that, it was refrigerated at 4°C to obtain fermented soybean cheese curd.

[0101] Among them, the soybean cheese starter with deodorizing and aroma-enhancing functions has a volume ratio of Pichia pastoris RS-13 and Pietrococcus FR23 bacterial suspensions of 1:1.

[0102] Comparative Example 1

[0103] Take 10 parts by weight of soy protein isolate, 100 parts by weight of water, 10 parts by weight of glucose, and 5.56 parts by weight of white sugar. Stir at 95°C and 500 r / min for 10 min. Then fill the container and sterilize it by heating in a water bath at 85°C for 10 min. Then cool it to 37°C to obtain unfermented soy cheese liquid.

[0104] Comparative Example 2

[0105] Take 10 parts by weight of soy protein isolate, 100 parts by weight of water, 10 parts by weight of glucose, and 5.56 parts by weight of white sugar. Stir at 95°C and 500 r / min for 10 min. Then fill the container and sterilize it by heating in a water bath at 85°C for 10 min. Then cool it to 37°C to obtain the soy cheese fermentation liquid.

[0106] Under sterile conditions, 6% (w / v) Chr. Hansen VEGA series plant-based cheese starter was inoculated into the soybean cheese fermentation liquid, fermented at 37°C for 8 hours, and then refrigerated at 4°C to obtain fermented soybean cheese curd.

[0107] Comparative Example 3

[0108] Take 10 parts by weight of soy protein isolate, 100 parts by weight of water, 10 parts by weight of glucose, and 5.56 parts by weight of white sugar. Stir at 95°C and 500 r / min for 10 min. Then fill the container and sterilize it by heating in a water bath at 85°C for 10 min. Then cool it to 37°C to obtain the soy cheese fermentation liquid.

[0109] Under sterile conditions, a bacterial suspension of 6% (v / v) Pediococcus lactis FR23 was inoculated into the fermentation liquid of soybean cheese, fermented at 37°C for 8 hours, and then refrigerated at 4°C to obtain fermented soybean cheese curd.

[0110] Comparative Example 4

[0111] Take 10 parts by weight of soy protein isolate, 100 parts by weight of water, 10 parts by weight of glucose, and 5.56 parts by weight of white sugar. Stir at 95°C and 500 r / min for 10 min. Then fill the container and sterilize it by heating in a water bath at 85°C for 10 min. Then cool it to 37°C to obtain the soy cheese fermentation liquid.

[0112] Under sterile conditions, a 6% (v / v) suspension of Pichia pastoris RS-13 was inoculated into the fermentation liquid of soybean cheese, fermented at 37°C for 8 hours, and then refrigerated at 4°C to obtain fermented soybean cheese curd.

[0113] Example 1

[0114] Sensory evaluations were performed on Examples 3-7 and Comparative Examples 1-4 above. Figure 2aExamples D1-D4 are Comparative Examples 1-4, and Examples D5-D9 are Examples 3-7. Significant differences (p<0.05) were found between the examples and comparative examples in aroma acceptability and overall acceptability, particularly in Examples 4 and 5, where aroma acceptability and overall acceptability were highest. Furthermore, the aroma sensory evaluation results ( Figure 2b From the perspective of sensory evaluation, different ratios of bacterial strains significantly affect the aroma, sourness, cucumber flavor, green aroma, milky aroma, sweet aroma, rice wine flavor, beany aroma, cheese flavor, and floral and fruity aroma of soybean cheese. For example, Comparative Examples 1 and 2 scored highly in beany aroma, soy flavor, and green aroma; Comparative Example 3 had a distinct cucumber and sour flavor; Comparative Example 4 had a distinct rice wine flavor; and Examples 3-7 had very low beany aroma and richer milky and fruity aromas. Based on the overall sensory evaluation results, when the ratio of Pichia pastoris to Pediococcus lactis was high, the fermented soybean cheese had the lowest beany aroma, the highest milky and floral / fruity aromas, and the best overall sensory quality. The GC-MS identification results are shown in Tables 1 and 2. Comparative Examples 1–4 (D1–D4) and Examples 3–7 (D5–D9) showed differences in some aroma components and their contents. Among them, 72 common aroma components were detected, including 18 alcohols, 25 aldehydes, 12 ketones, 4 acids, 4 esters, 3 furans, 3 phenols, and 2 ethers. Figure 3 There are 10 substances that give a beany smell, mainly aldehydes and furans. Figure 4 Compared to unfermented (D1) soybean cheese samples, fermentation with patented strains (D3-D9) reduced the beany flavor compounds, while commercially available plant-based cheese starter (D2) had no effect on reducing the beany flavor. Among the single-strain fermented samples, soybean cheese fermented with *Pediococcus lactis* (D3) showed a slight reduction in beany flavor compounds, but higher levels of milky and fruity aroma compounds such as 3-methylbutyraldehyde, hexanoic acid, methylheptenone, and 2-undecane. Soybean cheese fermented with *Pichia pastoris* (D4) had the lowest beany flavor compounds, but its aroma was relatively monotonous, consisting only of alcoholic compounds with a wine-like fragrance. Fermentation with a combination of strains (D6-D9) showed a reduction in beany flavor compounds and a significant increase in the content of alcohols, ketones, esters, and other fruity and milky aroma compounds. The combined fermentation also enriched the variety of volatile compounds. Figure 5 There were also differences between the different embodiments. When the ratio of Pichia pastoris to Pediococcus lactis was 50:1, the content of beany-smelling substances was the lowest, but the content of fruity, milky, and alcoholic flavorings, such as hexyl acetate, 3-methylbutyraldehyde, and 1-pentanol, was the highest, which is consistent with the sensory evaluation results. This may be due to a synergistic effect among these pleasant flavor compounds, masking the volatile beany flavor compounds.

[0115] Table 1. GC-MS identification results of volatile flavor compounds in soybean cheese

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[0121] Table 2. GC-MS identification results of volatile flavor compounds in soybean cheese.

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[0127] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.

Claims

1. A soybean cheese starter with deodorizing and aroma-enhancing properties, characterized in that, For containing fermented Pichia pastoris ( Pichia fermentans RS-13 and Pediococcus lactis ( Pediococcus acidilactici FR23 bacterial suspension; The preservation number of the fermented Pichia pastoris RS-13 is CCTCC NO: M 2020964, the depositary institution is China Center for Type Culture Collection, the deposit date is December 24, 2020, and the deposit address is Wuhan University, Wuhan, China. The accession number of the lactic acid cocci FR23 is CCTCC NO: M 20242058, the depositary institution is China Center for Type Culture Collection, the deposit date is September 24, 2024, and the deposit address is Wuhan University, Wuhan, China. The volume ratio of the bacterial suspension of Pichia pastoris RS-13 and the bacterial suspension of Pietrocarpus lactis FR23 is 50:

1. Removing the beany smell refers to reducing the production of substances that cause the beany smell, such as hexanal, benzaldehyde, 2-pentylfuran, and 1-octen-3-ol; enhancing the aroma refers to increasing the production of substances that cause the milky and fruity aroma, such as 3-methylbutanal, 2-nonanone, and hexyl acetate. The soybean cheese starter contains a total viable count of at least 1 × 10⁻⁶ Pichia pastoris RS-13 and Pediococcus lactis FR23. 9 CFU / mL.

2. The method for preparing the soybean cheese starter with deodorizing and aroma-enhancing properties as described in claim 1, characterized in that, Includes the following steps: Step 1: Streak the fermented Pichia pastoris RS-13 culture medium on YPD solid medium plates and streak the fermented Pichia pastoris FR23 culture medium on MRS solid medium plates until single colonies grow. Step 2: Inoculate the single colony of fermented Pichia pastoris RS-13 obtained in Step 1 into YPD liquid medium for liquid culture, and inoculate the single colony of lactic acid cocci FR23 obtained in Step 1 into MRS liquid medium for liquid culture, so as to obtain the culture medium of fermented Pichia pastoris RS-13 and the culture medium of lactic acid cocci FR23 respectively. Step 3: Centrifuge the culture medium of Pichia pastoris RS-13 and the culture medium of Pediococcus lactis FR23 separately, remove the supernatant, wash the cells with sterile water, add an equal volume of sterile water to the centrifuge tube, vortex to obtain the bacterial suspension of Pichia pastoris RS-13 and the bacterial suspension of Pediococcus lactis FR23 respectively. Step 4: Mix the bacterial suspension of Pichia pastoris RS-13 obtained in Step 3 with the bacterial suspension of Pietrocarpus lactis FR23 to obtain a soybean cheese starter with deodorizing and aroma-enhancing properties.

3. The method for preparing the soybean cheese starter with deodorizing and aroma-enhancing properties according to claim 2, characterized in that, The YPD solid culture medium consists of: 20.0 g / L peptone, 20.0 g / L glucose, 10.0 g / L yeast extract, and 13.5 g / L bacterial agar powder. The MRS solid culture medium consists of: 10.0 g / L peptone, 10.0 g / L beef meal, 5.0 g / L yeast extract, 20.0 g / L glucose, 0.1 g / L magnesium sulfate, 5.0 g / L sodium acetate, 2.0 g / L ammonium citrate, 2.0 g / L dipotassium hydrogen phosphate, 0.05 g / L manganese sulfate, 1.0 g / L Tween 80, and 13.5 g / L bacterial agar powder.

4. The method for preparing the soybean cheese starter with deodorizing and aroma-enhancing properties according to claim 2, characterized in that, The YPD liquid culture medium consists of: 20.0 g / L peptone, 20.0 g / L glucose, and 10.0 g / L yeast extract. The MRS liquid culture medium consists of: peptone 10.0 g / L, beef meal 10.0 g / L, yeast extract 5.0 g / L, glucose 20.0 g / L, magnesium sulfate 0.1 g / L, sodium acetate 5.0 g / L, ammonium citrate 2.0 g / L, dipotassium hydrogen phosphate 2.0 g / L, manganese sulfate 0.05 g / L, and Tween 80 1.0 g / L.

5. The preparation method of the soybean cheese fermentation agent with deodorizing and aroma-enhancing properties according to claim 2, characterized in that, In step 1, the conditions for streak incubation are: temperature 37 ℃, time 24~48 h; In step 2, the conditions for liquid culture are: temperature 37 ℃, time 10~15 h.

6. The method for preparing the soybean cheese starter with deodorizing and aroma-enhancing properties according to claim 2, characterized in that, In step 3, the centrifugation conditions are: temperature 4 ℃, rotation speed 10000 rpm, centrifugation time 5 min, and washing with sterile water 2-3 times; In step 3, the conditions for vortex oscillation are: vortex oscillation at 3000-6000 rpm for 5 minutes.

7. The application of the soybean cheese starter with deodorizing and aroma-enhancing properties as described in claim 1 in the preparation of fermented soybean cheese.

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