Soybean cheese leavening agent with fishy smell removing and aroma enhancing functional characteristics and application of soybean cheese leavening agent

Abstract

The invention relates to a soybean cheese leavening agent with fishy smell removing and aroma enhancing functional characteristics and an application of the soybean cheese leavening agent. The soybean cheese leavening agent disclosed by the invention comprises a fermented pichia pastoris RS-13 (the preservation number is CCTCC NO: M 2020964) and pediococcus acidilactici FR23 (the preservation number is CCTCC NO: M 20242058); according to the method, different strains are used for weakening the beany flavor and enhancing the flavor strength of milk flavor and fruit flavor, the variety, content and sensory evaluation results of key aroma components in the prepared fermented soybean cheese are used as main indexes, and the compounded strain combination can achieve the purposes of complementing the advantages of deodorization and aroma enhancement and optimizing the flavor. The soybean cheese prepared by the soybean cheese leavening agent has prominent milk flavor and fruit flavor, the beany flavor is reduced, the sensory performance is good and stable, the produced fermented soybean cheese product has better flavor, and the overall sensory acceptance and flavor quality of the fermented soybean cheese are improved.

Description

Technical Field

[0001] The invention belongs to the technical field of food fermentation, and in particular relates to a soybean cheese starter with the functional characteristics of removing fishy smell and enhancing flavor, and application thereof. Background Art

[0002] In recent years, consumers have become increasingly interested in plant-based cheeses, motivated by health, sustainability or animal welfare. Soybeans are widely available and high in protein, making them a high-quality raw material for the development of plant-based cheeses. However, soy products often have an unpleasant beany smell during processing, which directly affects the edible value of plant-based cheeses and limits their market development prospects. The beany smell of soy foods is mainly due to the activation of lipoxygenase in soybeans by oxygen and water when soybeans are crushed, which oxidizes the polyunsaturated fatty acids (mainly linoleic acid, linolenic acid, etc.) to form hydroperoxides, which are then degraded into a variety of volatile components such as low-molecular alcohols, aldehydes, ketones, acids and amines. Most of these small molecular compounds have different degrees of odor, thus forming a soybean smell, such as hexanal, benzaldehyde, 2-pentylfuran and 1-octen-3-ol.

[0003] Studies have shown that the beany smell of soybean products can be reduced through microbial fermentation. This mainly relies on the aroma production and enzymatic hydrolysis ability of specific microorganisms to produce metabolites with good flavor (such as 3-methylbutanal, 2-nonanone, and hexyl acetate) to mask the beany smell, or to degrade and transform the beany smell substances, thereby achieving the effect of reducing the beany smell and improving the overall acceptance of the sample.

[0004] Chinese patent CN113151018B discloses a fermented Pichia RS-13 and its application, with a deposit number of CCTCCNO: M 2020964, a Latin name of Pichia fermentans RS-13, and was deposited in the China Center for Type Culture Collection on December 24, 2020. Experiments have shown that after fermenting okara, the fermented Pichia can reduce the beany smell in the okara to a level that is imperceptible to the human body, and the content of beany smell compounds is reduced to 1 / 40 of that of unfermented okara; fermented Pichia RS-13 is used in okara beverage products to prepare okara beverages without beany smell, which can effectively solve the technical problems of low utilization rate of okara due to severe bean smell and limited development of related products, and has good application prospects.

[0005] Chinese patent CN116982659A discloses a low beany pea protein plant cheese and its preparation method. The pea protein plant cheese provided by it is obtained by dry-heat mixing pea protein powder and potato starch in proportion to obtain a mixed powder with reduced beany smell once, and then adding the mixed powder to the oil-water mixture prepared in proportion, and then adding auxiliary materials such as thickener and emulsifier and heating and stirring until all the mixed materials are fine and smooth mucus, access to Pichia yeast fermentation, and stop fermentation when the fermentation end pH reaches 5.0-5.2, pour into the mold and refrigerate to obtain pea protein plant cheese with secondary beany smell reduction. It simultaneously uses the technology of dry heat mixing and solid-state fermentation to achieve the removal of the beany smell of pea protein plant cheese. Compared with ordinary pea protein plant cheese in flavor characteristics, the beany smell is significantly reduced and the fermentation flavor is enhanced.

[0006] However, both of the above patents use Pichia fermentans RS-13 alone to reduce the beany smell.

[0007] At present, most dairy fermentation agents have been optimized in milk matrices, but are not suitable for non-dairy matrices. The bacteria used for fermentation in soy products and that can reduce the beany smell are mostly yeasts, lactic acid bacteria and molds. Their single-bacteria fermentation has some shortcomings, but there are few studies on the use of combined strain fermentation to reduce the beany smell of soy products, and the addition ratio of the combined strains also needs further exploration. Summary of the invention

[0008] The technical problems to be solved by the present invention are: soybean cheese has a serious beany smell and lacks flavor compared with traditional cheese; existing cheese starter is not suitable for plant-based cheese matrix; and single-strain fermentation has insufficient flavor.

[0009] In order to solve the above technical problems, the present invention provides a soybean cheese starter with the functional characteristics of removing fishy smell and enhancing flavor, and application thereof.

[0010] The purpose of the present invention is achieved through the following technical solutions:

[0011] The present invention provides a soybean cheese starter with the functional characteristics of removing fishy smell and enhancing flavor, which is a bacterial suspension containing Pichia pastoris RS-13 and Pediococcus acidilactici FR23;

[0012] The Pichia fermentans RS-13 has a deposit number of CCTCC NO: M2020964, the deposit unit is China Center for Type Culture Collection, the deposit time is December 24, 2020, and the deposit address is Wuhan University, Wuhan, China;

[0013] The preservation number of the Pediococcus acidilactici FR23 is CCTCC NO: M20242058, the preservation unit is the China Center for Type Culture Collection, the preservation time is September 24, 2024, and the preservation address is Wuhan University, Wuhan, China.

[0014] In one embodiment of the present invention, the total viable count of Pichia pastoris RS-13 and Pediococcus acidilactici FR23 in the soy cheese starter is at least 1×10 9 CFU / mL.

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

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

[0017] The present invention further provides the use of Pichia pastoris RS-13 or Pediococcus acidilactici FR23 in preparing a soybean cheese starter, and the prepared soybean cheese starter has the functional characteristics of removing fishy smell and enhancing flavor.

[0018] The present invention further provides a method for preparing the soybean cheese starter with the functional characteristics of removing fishy smell and enhancing flavor, comprising the following steps:

[0019] Step 1: streak the bacterial solution of Pichia pastoris RS-13 on a YPD solid culture medium plate, and streak the bacterial solution of Pediococcus acidilactici FR23 on an MRS solid culture medium plate until a single colony grows;

[0020] Step 2: inoculating a single colony of Pichia pastoris RS-13 obtained in step 1 into a liquid culture medium containing YPD broth for liquid culture, and inoculating a single colony of Pediococcus acidilactici FR23 obtained in step 1 into a liquid culture medium containing Pediococcus acidilactici FR23 broth for liquid culture, to obtain a culture solution of Pichia pastoris RS-13 and a culture solution of Pediococcus acidilactici FR23, respectively;

[0021] Step 3: centrifuging the culture broth of Pichia pastoris RS-13 and the culture broth of Pediococcus acidilactici FR23 respectively, removing the supernatant, washing the bacteria with sterile water, then adding an equal volume of sterile water to the centrifuge tube, vortexing and shaking, to obtain bacterial suspensions of Pichia pastoris RS-13 and Pediococcus acidilactici FR23 respectively;

[0022] Step 4: Mix the bacterial suspension of Pichia pastoris RS-13 and the bacterial suspension of Pediococcus acidilactici FR23 obtained in step 3 to obtain a soybean cheese starter with the functional characteristics of removing fishy smell and enhancing flavor.

[0023] In a specific embodiment of the present invention, the method for preparing the soybean cheese starter with the functional characteristics of removing fishy smell and enhancing flavor comprises the following steps:

[0024] Step 1: taking out the preserved Pichia pastoris RS-13 and Pediococcus acidilactici FR23 bacterial liquids from the glycerol preservation tubes respectively, using an inoculation loop to streak the Pichia pastoris RS-13 bacterial liquid on a YPD solid medium plate, using an inoculation loop to streak the Pediococcus acidilactici FR23 bacterial liquid on an MRS solid medium plate, and then culturing them at a constant temperature in an incubator respectively until a single colony grows;

[0025] Step 2: Pick a single colony of each 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 media, respectively, and culture them in a constant temperature incubator to obtain culture solutions of the two strains Pichia pastoris RS-13 and Pediococcus acidilactici FR23, respectively;

[0026] Step 3: centrifuge the culture broths of the two strains Pichia pastoris RS-13 and Pediococcus acidilactici FR23 respectively, remove the supernatant, wash the bacteria with sterile water, then add an equal volume of sterile water to the centrifuge tube, and vortex to obtain a bacterial suspension of the two strains;

[0027] Step 4: Mix the two bacterial suspensions obtained in step 3 according to a certain proportion to obtain a soybean cheese starter with the functions of removing fishy smell and enhancing flavor.

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

[0029] In one embodiment of the present invention, the components of the MRS solid culture medium are: 10.0 g / L peptone, 10.0 g / L beef powder, 5.0 g / L yeast powder, 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 streaking culture conditions are: temperature 37° C., time 24 to 48 hours.

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

[0032] In one embodiment of the present invention, in step 2, the components of the MRS liquid medium are: peptone 10.0 g / L, beef powder 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, potassium dihydrogen phosphate 2.0 g / L, manganese sulfate 0.05 g / L, Tween 80 1.0 g / L.

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

[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 vortexing condition is: vortexing at 3000-6000 rpm for 5 min.

[0036] The present invention further provides the use of the soybean cheese starter with the functional characteristics of removing fishy smell and enhancing flavor in the preparation of fermented soybean cheese.

[0037] Compared with the prior art, the present invention has the following beneficial effects:

[0038] The soybean cheese fermentation agent with the function of removing fishy smell and enhancing flavor of the present invention is compounded by Pichia pastoris RS-13 and Pediococcus acidilactici FR23. The present invention has found that Pediococcus acidilactici FR23 has the function of removing fishy smell and enhancing flavor, and then compounding Pichia pastoris RS-13 and Pediococcus acidilactici FR23 can make the content of beany smell substances lower after compounding, and their flavoring effects are different, which can make the aroma richer. The soybean cheese fermented by the above two bacteria has lower content of beany smell substances such as hexanal, benzaldehyde, 2-pentylfuran and 1-octen-3-ol, and higher content of milky and fruity smell substances such as 3-methylbutanal, 2-nonanone and hexyl acetate.

[0039] The present invention utilizes the complementary performance of different strains in reducing beany smell and enhancing milky and fruity flavor intensity, and takes the types, contents and sensory evaluation results of key aroma components in the produced fermented soybean cheese as main indicators. The purpose of achieving the purpose of complementary advantages of removing fishy smell and enhancing flavor and optimizing flavor by using the composite strain combination can be achieved.

[0040] The soybean cheese prepared by the soybean cheese starter of the present invention has prominent milk flavor and fruity flavor, reduced beany smell, good and stable sensory performance, and the fermented soybean cheese product produced thereby has better flavor, which improves the overall sensory acceptance and flavor quality of the fermented soybean cheese. The present application not only solves the flavor problem in the development process of plant-based foods by endogenous flavor enhancement, but also meets the current consumer's pursuit of green and healthy foods, and conforms to the trend of "clean label". BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 This is a process flow chart for soy cheese fermentation;

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

[0043] Figure 2(b) is a radar chart of the aroma sensory evaluation results of different soy cheeses;

[0044] Figure 3 The stacked bar graph of volatile flavor substances in soybean cheese fermented with different bacterial strains and ratios;

[0045] Figure 4 The heat map of volatile flavor compounds in soy cheese fermented with different bacterial strains and ratios;

[0046] Figure 5 Changes in the content of aroma substances in soy cheese fermented with different strains of bacteria.

[0047] Collection Instructions

[0048] The Pichia fermentans RS-13 has a deposit number of CCTCC NO: M2020964, the deposit unit is China Center for Type Culture Collection, the deposit time is December 24, 2020, and the deposit address is Wuhan University, Wuhan, China;

[0049] The preservation number of the Pediococcus acidilactici FR23 is CCTCC NO: M20242058, the preservation unit is the China Center for Type Culture Collection, the preservation time is September 24, 2024, and the preservation address is Wuhan University, Wuhan, China. DETAILED DESCRIPTION

[0050] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

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

[0052] The components of the MRS solid culture medium used in each embodiment of the present invention are: peptone 10.0 g / L, beef powder 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, potassium dihydrogen phosphate 2.0 g / L, manganese sulfate 0.05 g / L, Tween 80 1.0 g / L and bacterial agar powder 13.5 g / L.

[0053] The components of the YPD liquid culture medium used in each embodiment of the present invention are: 20.0 g / L peptone, 20.0 g / L glucose, and 10.0 g / L yeast extract powder.

[0054] The components of the MRS liquid culture medium used in each embodiment of the present invention are: peptone 10.0 g / L, beef powder 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, potassium dihydrogen phosphate 2.0 g / L, manganese sulfate 0.05 g / L, Tween 80 1.0 g / L.

[0055] The aroma compounds in the fermented soybean cheese obtained in each embodiment of the present invention are determined by gas chromatography-mass spectrometry (GC-MS) in the following steps:

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

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

[0058] Example 1

[0059] Pichia fermentans RS-13, the strain was isolated from the traditional Chinese fermented food, milk fan, by the Food Flavor Control Innovation Team of the School of Flavor and Fragrance Technology and Engineering of Shanghai University of Technology, and is preserved in the China Center for Type Culture Collection, the preservation address: Wuhan University, Wuhan, China, postal code: 430072, the preservation number is (CCTCCM2020964). The isolation and screening of Pichia fermentans RS-13 is recorded in patent CN113151018A.

[0060] The isolation and screening of strain FR23 was carried out as follows:

[0061] (1) Under sterile conditions, 1 g of sample (the sample is Guizhou fermented bean curd, which is naturally fermented locally and stored in a 4°C refrigerator until sampling) is taken into a sterile test tube, 9 ml of physiological saline is added to the sterile test tube, and mixed to obtain a dilution of 10 -1 of bacterial liquid;

[0062] (2) Take the dilution obtained in step (1) as 10 -1 Place 1 mL of bacterial solution in another sterile test tube, add 9 mL of saline to the test tube, and mix to obtain a dilution gradient of 10 -2 Repeat the above steps 4 times to obtain 10 -3 , 10 -4 , 10 -5 and 10 -6 Four gradients of bacterial solution;

[0063] (3) From step (2), 10 -3 ~10 -6 0.1 mL of bacterial solution from the four gradients was spread on MRS solid medium plates and placed in an inverted culture in a 37°C incubator for 24-48 h.

[0064] (4) using a sterilized toothpick to pick up a single colony of the isolated strain obtained after plate culture in step (3), inoculating it into MRS liquid culture medium, and culturing it in a 37° C. incubator for 12 h to obtain a fermentation broth of the isolated strain;

[0065] (5) The fermentation broth of the isolated strain corresponding to step (4) was centrifuged at 10,000 rpm for 5 min at 4° C., the supernatant was discarded after centrifugation, an equal volume of sterile water was added, and the mixture was centrifuged at 10,000 rpm for 5 min, the supernatant was discarded, and the mixture was washed 2-3 times in this manner. Finally, an equal volume of sterile water was added and the mixture was vortexed at 6,000 rpm for 5 min to prepare a bacterial suspension for later use;

[0066] (6) 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 are taken, stirred at 95° C. and 500 r / min for 10 min; then filled, sterilized by heating in a water bath at 85° C. for 10 min, and then cooled to 37° C. to obtain a sterile soy cheese inoculation solution;

[0067] (7) taking a portion of the bacterial suspension in step (5) and another portion of the bacterial suspension prepared by Pichia fermentans RS-13, inoculating the soy cheese inoculation solution in (6) with the bacterial suspension at an inoculum amount of 6% (v / v) under a sterile environment, fermenting at 37° C. for 8 h, and then refrigerating at 4° C. to obtain fermented soy cheese curd;

[0068] The above two kinds of fermented soybean cheese were subjected to sensory evaluation and GC-MS identification. The experimental results showed that both strains had the effect of removing fishy smell. The fermented soybean cheese made by strain RS-13 had almost no beany smell, and also produced some alcoholic substances such as isopentanol and phenylethanol, but the overall flavor was relatively bland and the curdling was poor; the fermented soybean cheese made by strain FR23 had a richer aroma, with more obvious milky and fruity aromas, such as higher content of 3-methylbutanal and 2,3-butanediol, and the beany smell was lower than that of the unfermented one, but it could also be clearly perceived. Finally, these two strains with the function of removing fishy smell and enhancing flavor were selected to compound fermented soybean cheese.

[0069] The strain FR23 was biologically identified, including PCR amplification of genomic DNA and 16S rRNA sequence detection; the identification results of strain RS-13 are recorded in patent CN113151018A. The primers used for PCR amplification were 27F (5'-GAGAGTTTGATCCTGGCTCAG-3') (SEQ ID NO: 1) and 1492r (5'-TACGGCTACCTTGTTACGAC-3') (SEQID NO: 2), and the amplification system was: 2 μL of 27F primer, 2 μL of 1492r primer, 2 μL of bacterial solution, 19 μL of ddH2O and 25 μL of 2×Gold Star Best Mixed. The PCR reaction procedure was: (1) 95°C / 10 min, (2) 94°C / 30 s, (3) 55°C / 30 s, (4) 72°C / 1.5 min, steps (2) to (4) were repeated for 30 cycles, (5) 72°C / 5 min, and (6) stored at 4°C. After PCR amplification, agarose gel electrophoresis was performed. The presence of clear bands in the measured fragments on the gel imager indicated that the PCR was successful. The remaining bacterial samples of 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 sent to the China Center for Type Culture Collection for preservation, with the preservation number (CCTCC M 20242058).

[0070] Example 2

[0071] A soybean cheese starter with the function of removing fishy smell and enhancing flavor 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 comprises the following steps:

[0073] (1) The bacterial liquids of Pichia fermentans RS-13 and Pediococcus acidilactici FR23 were taken out from the glycerol storage tubes respectively, and the bacterial liquid of Pichia fermentans RS-13 was streaked on a YPD solid medium plate with an inoculation loop, and the bacterial liquid of Pediococcus acidilactici FR23 was streaked on an MRS solid medium plate with an inoculation loop, and then placed in a 37°C incubator for 24 hours until a single colony grew;

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

[0075] (3) The culture broths of the two strains obtained in (2) were centrifuged at 10,000 rpm for 5 min at 4°C, and the supernatant was discarded. An equal volume of sterile water was added, and the culture broths were centrifuged at 10,000 rpm for 5 min. The supernatant was discarded. This was repeated 2 to 3 times. Finally, an equal volume of sterile water was added, and the culture broths were vortexed at 6,000 rpm for 5 min to prepare a bacterial suspension for use. The bacterial content in the bacterial suspension was 1 × 10 9 CFU / mL;

[0076] (4) The bacterial suspensions of Pichia pastoris RS-13 and Pediococcus acidilactici FR23 obtained in (3) were mixed at a volume ratio of 1:1 to 100:1 to obtain a soybean cheese starter with the function of removing fishy smell and enhancing flavor, wherein the total number of viable bacteria in the starter was 1×10 9 CFU / mL.

[0077] Example 3

[0078] The application of the soybean cheese starter with the function of removing fishy smell and enhancing flavor obtained in Example 2 in fermenting soybean cheese specifically comprises the following steps: Figure 1 The soy cheese fermentation process shown below:

[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, and stir at 95°C at a speed of 500 r / min for 10 minutes; then fill, and then sterilize by heating in a water bath at 85°C for 10 minutes, and then cool to 37°C to obtain a soy cheese fermentation liquid.

[0080] In a sterile environment, the soybean cheese fermentation liquid was inoculated with the soybean cheese starter described in Example 2 at a total inoculation amount of 6% (v / v), fermented at 37° C. for 8 h, and then refrigerated at 4° C. to obtain fermented soybean cheese curd.

[0081] Among them, the volume ratio of the bacterial suspension of Pichia pastoris RS-13 and Pediococcus acidilactici FR23 with the function of removing fishy smell and enhancing flavor of soybean cheese starter was 100:1, and the bacterial content in the bacterial suspension was 1×10 9 CFU / mL.

[0082] Example 4

[0083] The application of the soybean cheese starter with the function of removing fishy smell and enhancing flavor obtained in Example 2 in fermenting soybean cheese specifically comprises 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, and stir at 95°C at a speed of 500 r / min for 10 minutes; then fill, and then sterilize by heating in a water bath at 85°C for 10 minutes, and then cool to 37°C to obtain a soy cheese fermentation liquid.

[0085] In a sterile environment, the soybean cheese fermentation liquid was inoculated with the soybean cheese starter described in Example 2 at a total inoculation amount of 6% (v / v), fermented at 37° C. for 8 h, and then refrigerated at 4° C. to obtain fermented soybean cheese curd.

[0086] Among them, the volume ratio of the bacterial suspension of Pichia pastoris RS-13 and Pediococcus acidilactici FR23 in the soybean cheese starter with the function of removing fishy smell and enhancing flavor is 50:1.

[0087] Example 5

[0088] The application of the soybean cheese starter with the function of removing fishy smell and enhancing flavor obtained in Example 2 in fermenting soybean cheese specifically comprises 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, and stir at 95°C at a speed of 500 r / min for 10 minutes; then fill, and then sterilize by heating in a water bath at 85°C for 10 minutes, and then cool to 37°C to obtain a soy cheese fermentation liquid.

[0090] In a sterile environment, the soybean cheese fermentation liquid was inoculated with the soybean cheese starter described in Example 2 at a total inoculation amount of 6% (v / v), fermented at 37° C. for 8 h, and then refrigerated at 4° C. to obtain fermented soybean cheese curd.

[0091] Among them, the volume ratio of the bacterial suspension of Pichia pastoris RS-13 and Pediococcus acidilactici FR23 in the soybean cheese starter with the function of removing fishy smell and enhancing flavor is 10:1.

[0092] Example 6

[0093] The application of the soybean cheese starter with the function of removing fishy smell and enhancing flavor obtained in Example 2 in fermenting soybean cheese specifically comprises 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, and stir at 95°C at a speed of 500 r / min for 10 minutes; then fill, and then sterilize by heating in a water bath at 85°C for 10 minutes, and then cool to 37°C to obtain a soy cheese fermentation liquid.

[0095] In a sterile environment, the soybean cheese fermentation liquid was inoculated with the soybean cheese starter described in Example 2 at a total inoculation amount of 6% (v / v), fermented at 37° C. for 8 h, and then refrigerated at 4° C. to obtain fermented soybean cheese curd.

[0096] Among them, the volume ratio of the bacterial suspension of Pichia pastoris RS-13 and Pediococcus acidilactici FR23 in the soybean cheese starter with the function of removing fishy smell and enhancing flavor is 5:1.

[0097] Example 7

[0098] The application of the soybean cheese starter with the function of removing fishy smell and enhancing flavor obtained in Example 2 in fermenting soybean cheese specifically comprises 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, and stir at 95°C at a speed of 500 r / min for 10 minutes; then fill, and then sterilize by heating in a water bath at 85°C for 10 minutes, and then cool to 37°C to obtain a soy cheese fermentation liquid.

[0100] In a sterile environment, the soybean cheese fermentation liquid was inoculated with the soybean cheese starter described in Example 2 at a total inoculation amount of 6% (v / v), fermented at 37° C. for 8 h, and then refrigerated at 4° C. to obtain fermented soybean cheese curd.

[0101] Among them, the volume ratio of the bacterial suspension of Pichia pastoris RS-13 and Pediococcus acidilactici FR23 in the soybean cheese starter with the function of removing fishy smell and enhancing flavor is 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 granulated sugar, and stir at 95°C and a speed of 500 r / min for 10 minutes; then fill, and then sterilize by heating in a water bath at 85°C for 10 minutes, and then cool to 37°C to obtain an unfermented soy cheese stock solution.

[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, and stir at 95°C at a speed of 500 r / min for 10 minutes; then fill, and then sterilize by heating in a water bath at 85°C for 10 minutes, and then cool to 37°C to obtain a soy cheese fermentation liquid.

[0106] In a sterile environment, 6% (w / v) of Chr. Hansen's VEGA series plant-based cheese starter was inoculated into the soy cheese fermentation liquid, fermented at 37°C for 8 hours, and then refrigerated at 4°C to obtain fermented soy 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, and stir at 95°C at a speed of 500 r / min for 10 minutes; then fill, and then sterilize by heating in a water bath at 85°C for 10 minutes, and then cool to 37°C to obtain a soy cheese fermentation liquid.

[0109] In a sterile environment, 6% (v / v) bacterial suspension of Pediococcus acidilactici FR23 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.

[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, and stir at 95°C at a speed of 500 r / min for 10 minutes; then fill, and then sterilize by heating in a water bath at 85°C for 10 minutes, and then cool to 37°C to obtain a soy cheese fermentation liquid.

[0112] In a sterile environment, 6% (v / v) bacterial suspension of Pichia pastoris RS-13 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.

[0113] Effect Example 1

[0114] Sensory evaluation was performed on the above Examples 3 to 7 and Comparative Examples 1 to 4 ( Figure 2a), D1-D4 are comparative examples 1 to 4, and D5-D9 are examples 3 to 7. There are significant differences (p<0.05) between the examples and the comparative examples in terms of aroma acceptance and overall acceptance, especially examples 4 and 5 have the highest aroma acceptance and overall acceptance. In addition, from the results of aroma sensory evaluation ( Figure 2b ), different bacterial species compounding ratios will have a significant impact on the bean flavor, sourness, cucumber flavor, green flavor, milk flavor, sweet flavor, rice wine flavor, beany flavor, cheese flavor, and floral and fruity flavor of soybean cheese. For example, comparative examples 1 and 2 have high scores for beany flavor, bean flavor, and green flavor, comparative example 3 has obvious cucumber flavor and sourness, comparative example 4 has obvious rice wine flavor, and examples 3 to 7 have very low beany flavor, and have richer milk flavor, fruity flavor, etc. From the results of comprehensive sensory evaluation, when the compounding ratio of Pichia pastoris and Pediococcus acidilactici is high, the fermented soybean cheese has the lowest beany flavor, the highest milk flavor and floral and fruity flavor, and the best overall sensory experience. The GC-MS identification results are shown in Tables 1 and 2. Comparative Examples 1 to 4 (D1-D4) and Examples 3 to 7 (D5-D9) differ in some aroma components and contents; 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 beany smell substances, mainly aldehydes and furans ( Figure 4 ). Compared with the unfermented (D1) soy cheese samples, the beany odor substances were reduced after fermentation with the patented strain (D3-D9), while the commercially available plant-based cheese starter (D2) had no effect on reducing the beany odor. Among the samples fermented with single bacteria, the beany odor substances in the soy cheese fermented with Pediococcus acidilactici (D3) were slightly reduced, but the content of substances with milky, floral and fruity aromas such as 3-methylbutanal, hexanoic acid, methylheptenone, and 2-undecanone was relatively high; the soy cheese fermented with Pichia pastoris (D4) had the lowest beany odor substances, but the aroma was relatively monotonous, with only alcohol substances with wine aroma. Through the composite fermentation of bacterial strains (D6-D9), it can be seen that the content of beany odor substances is reduced, and the content of floral, fruity and milky aroma substances such as alcohols, ketones and esters is significantly increased. The composite fermentation of bacterial strains also enriches the types of volatile compounds ( Figure 5 ). There are also differences between different embodiments. When the ratio of Pichia pastoris to Pediococcus acidilactici is 50:1, the beany smell substance content is the lowest, but the content of substances with fruity, milky and mellow aromas such as hexyl acetate, 3-methylbutyraldehyde and 1-pentanol is the highest, which is consistent with the sensory evaluation results. This may be due to the synergistic effect between these pleasant flavor substances, and the beany volatile flavor substances are masked.

[0115] Table 1 Identification results of volatile flavor compounds in soy cheese by GC-MS

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[0119]

[0120]

[0121] Table 2 Identification results of volatile flavor compounds in soy cheese by GC-MS

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[0125]

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[0127] The above description of the embodiments is to facilitate the understanding and use of the invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments, and 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 present invention should be within the scope of protection of the present invention.

Claims

1. A soybean cheese starter with the functional characteristics of removing fishy smell and enhancing flavor, characterized in that: A bacterial suspension comprising Pichia pastoris RS-13 and Pediococcus acidilactici FR23; The Pichia fermentans RS-13 has a deposit number of CCTCC NO: M2020964, the deposit unit is China Center for Type Culture Collection, the deposit time is December 24, 2020, and the deposit address is Wuhan University, Wuhan, China; The preservation number of the Pediococcus acidilactici FR23 FR23 is CCTCCNO: M 20242058, the preservation unit is the China Center for Type Culture Collection, the preservation time is September 24, 2024, and the preservation address is Wuhan University, Wuhan, China.

2. The soybean cheese starter with the functional characteristics of removing fishy smell and enhancing flavor according to claim 1, characterized in that: The total number of viable bacteria of Pichia pastoris RS-13 and Pediococcus acidilactici FR23 contained in the soybean cheese starter is at least 1×10 9 CFU / mL.

3. The soybean cheese starter with the functional characteristics of removing fishy smell and enhancing flavor according to claim 1, characterized in that: The soybean cheese starter is composed of a mixture of a bacterial suspension of Pichia pastoris RS-13 and a bacterial suspension of Pediococcus acidilactici FR23, wherein the volume ratio of the bacterial suspension of Pichia pastoris RS-13 to the bacterial suspension of Pediococcus acidilactici FR23 is 1:1 to 100:

1.

4. Application of Pediococcus acidilactici FR23 in the preparation of soybean cheese starter, characterized in that: The prepared soybean cheese starter has the functional characteristics of removing fishy smell and enhancing flavor. The preservation number of the Pediococcus acidilactici FR23 is CCTCC NO: M20242058, the preservation unit is the China Center for Type Culture Collection, the preservation time is September 24, 2024, and the preservation address is Wuhan University, Wuhan, China.

5. The method for preparing the soybean cheese starter with the functional characteristics of removing fishy smell and enhancing flavor according to any one of claims 1 to 3, characterized in that: The following steps are involved: Step 1: streak the bacterial solution of Pichia pastoris RS-13 on a YPD solid culture medium plate, and streak the bacterial solution of Pediococcus acidilactici FR23 on an MRS solid culture medium plate until a single colony grows; Step 2: inoculating a single colony of Pichia pastoris RS-13 obtained in step 1 into a liquid culture medium containing YPD broth for liquid culture, and inoculating a single colony of Pediococcus acidilactici FR23 obtained in step 1 into a liquid culture medium containing Pediococcus acidilactici FR23 broth for liquid culture, to obtain a culture solution of Pichia pastoris RS-13 and a culture solution of Pediococcus acidilactici FR23, respectively; Step 3: centrifuging the culture broth of Pichia pastoris RS-13 and the culture broth of Pediococcus acidilactici FR23 respectively, removing the supernatant, washing the bacteria with sterile water, then adding an equal volume of sterile water to the centrifuge tube, vortexing and shaking, to obtain bacterial suspensions of Pichia pastoris RS-13 and Pediococcus acidilactici FR23 respectively; Step 4: Mix the bacterial suspension of Pichia pastoris RS-13 and the bacterial suspension of Pediococcus acidilactici FR23 obtained in step 3 to obtain a soybean cheese starter with the functional characteristics of removing fishy smell and enhancing flavor.

6. The method for preparing the soybean cheese starter with the functional characteristics of removing fishy smell and enhancing flavor according to claim 5, characterized in that: The YPD solid culture medium comprises: 20.0 g / L peptone, 20.0 g / L glucose, 10.0 g / L yeast extract powder and 13.5 g / L bacterial agar powder; The components of the MRS solid culture medium are: 10.0 g / L peptone, 10.0 g / L beef powder, 5.0 g / L yeast powder, 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.

7. The method for preparing the soybean cheese starter with the functional characteristics of removing fishy smell and enhancing flavor according to claim 5, characterized in that: The components of the YPD liquid culture medium are: 20.0 g / L peptone, 20.0 g / L glucose, and 10.0 g / L yeast extract powder; The components of MRS liquid culture medium are: peptone 10.0 g / L, beef powder 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, Tween 80 1.0 g / L.

8. The method for preparing the soybean cheese starter with the functional characteristics of removing fishy smell and enhancing flavor according to claim 5, characterized in that: In step 1, the streak culture conditions are: temperature 37°C, time 24 to 48 hours; In step 2, the conditions of the liquid culture are: temperature 37° C., time 10 to 15 hours.

9. The method for preparing the soybean cheese starter with the functional characteristics of removing fishy smell and enhancing flavor according to claim 5, characterized in that: 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; In step 3, the vortexing conditions are: vortexing at 3000-6000 rpm for 5 min.

10. Use of the soybean cheese starter with the functional characteristics of removing fishy smell and enhancing flavor according to any one of claims 1 to 3 in preparing fermented soybean cheese.

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