CN108740062B - Preparation method of oil fermented bean curd with high flowery flavor and fruit flavor yield - Google Patents
Preparation method of oil fermented bean curd with high flowery flavor and fruit flavor yield Download PDFInfo
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
- A23C20/00—Cheese substitutes
- A23C20/02—Cheese substitutes containing neither milk components, nor caseinate, nor lactose, as sources of fats, proteins or carbohydrates
- A23C20/025—Cheese substitutes containing neither milk components, nor caseinate, nor lactose, as sources of fats, proteins or carbohydrates mainly containing proteins from pulses or oilseeds
Abstract
The invention discloses a preparation method of high-yield flower fragrance and fruity oil fermented bean curd, and belongs to the technical field of bean product fermentation. The Mucor racemosus spore suspension is sprayed on the surface of the sour bean curd for pre-fermentation, wherein the Mucor racemosus is the Mucor racemosus with the preservation number of CGMCC No.15264, the strains have high capability of producing linalool and linalyl acetate, and 9645.14 mu g/kg of linalool and 1228.05 mu g/kg of linalyl acetate can be produced after 90 days of post-fermentation, so that the prepared fermented bean curd has flower fragrance and fruit fragrance; and the production process of the oil fermented bean curd is pure bacteria fermentation, and the controllability is remarkable, so that the possibility of over fermentation is reduced, and the mould taste of the final product is not remarkable.
Description
Technical Field
The invention relates to a preparation method of oil fermented bean curd with high flower fragrance and fruit fragrance, and belongs to the technical field of bean product fermentation.
Background
The fermented bean curd is a seasoning and table product prepared by processing and grinding soybeans as main raw materials, making blanks, cultivating bacteria and fermenting. The traditional fermented bean product with national characteristics in China has the characteristics of rich nutrition, fine and smooth texture, delicious taste and appetitive attraction, is a favorite appetizing appetizer and condiment, and occupies an important position in the fermented bean product.
Linalool has fragrance of flower fragrance and contributes greatly to the fragrance of fermented bean curd; research shows that the content of linalool in the red fermented bean curd is 738.24 mu g/kg, which has great contribution to the aroma of the red fermented bean curd (dawn red, high willow, Dang Fei, and the like. identification of characteristic aroma components of red fermented bean curd fermented by mixed strains [ J ] food and fermentation industry, 2017,43(2): 185-. Organic acid and alcohol produced by the synergistic metabolism of microorganisms such as mucor and saccharomycetes in the fermented bean curd form various esters to form the special fragrance of the fermented bean curd (Chengchang. the separation, identification, fermentation performance and mycelium autolysis research of the mucor of the fermented bean curd [ D ]. Guiyang: Guizhou university, 2008). Studies show that the contents of linalyl acetate are 641.2 μ g/kg and 527.8 μ g/kg respectively when extracting and analyzing volatile components from whole fermented bean curd and traditional fermented bean curd (leather, leather. Korea. headspace solid phase microextraction-gas chromatography coupled technology is used for analyzing volatile components [ J ]. Chinese seasoning, 2014, 39 (12): 105-. In the paper, "research on red bean curd fermentation and volatile flavor components thereof", flavor analysis was performed on red bean curd and 3 commercially available oil fermented beancurd of different brands, wherein the content of linalool in 4 fermented beancurd is 303 μ g/kg, 321 μ g/kg, 115 μ g/kg and 200 μ g/kg in sequence, and the content of linalyl acetate is 0 μ g/kg, 32 μ g/kg, 0 μ g/kg and 16 μ g/kg in sequence (Lujing. research on red bean curd fermentation and volatile flavor components thereof [ D ]. Chengdu: Xihua university, 2015).
The special geographical and climatic conditions in the Yunnan mu oding area enable a unique microbial flora which is beneficial to the fermentation of the oil fermented bean curd to be formed in the area, so that the produced oil fermented bean curd has excellent quality and rich flower fragrance and fruit fragrance. However, the production of the fermented bean curd in the Yunnan muggen area adopts natural fermentation, so that the pollution of mixed bacteria is serious, and the fragrance of the product is unstable.
Disclosure of Invention
In order to solve the existing problems, the invention provides a preparation method of oil fermented bean curd with high flower and fruit fragrance yield, and the technical scheme is as follows: a preparation method of oil fermented bean curd with high flower fragrance and fruit fragrance comprises the steps of taking sour bean curd as a raw material, spraying Mucor racemosus spore suspension to the surface of the sour bean curd for pre-fermentation, airing and salting, adding seasonings, and performing post-fermentation; wherein the Mucor racemosus is preserved in the China general microbiological culture Collection center in 2018, month 01 and day 18, and the preservation numbers are as follows: CGMCC No.15264, the preservation address is No. 3 of the institute of microbiology of the national academy of sciences of China, institute of Xilu No.1 of Beijing, Chaoyang.
Mucor racemosus (Mucor racemosus) CGMCC No.15264 has long mycelium and white color.
Optionally, the sour bean curd is obtained by inoculating lactobacillus into soybean milk and gelling for 3-5 hours; the preparation steps are as follows: stirring and crushing the prepared soybean curd, then pouring the soybean curd into a bean curd forming groove, and pressing and forming.
Optionally, the concentration of the total mucor spore suspension is 100-110/mL.
Optionally, the airing time is 10-14 hours, and the salting parameter is adding 5% of salt, salting for 12-14 hours, and removing 12-18% of water.
Optionally, the seasoning is added for post-fermentation, namely, the seasoning and the fermented bean curd blank are uniformly mixed, 12 blocks are put into a glass bottle, 100mL of rapeseed oil is added, and the post-fermentation is carried out for 90 days.
Optionally, the seasoning comprises pepper and zanthoxylum.
Optionally, after the press forming, the method further comprises: the bean curd was cut into 3.5cm by 2.0 cm.
The invention has the beneficial effects that:
the Mucor racemosus spore suspension is sprayed on the surface of the sour bean curd for pre-fermentation, wherein the Mucor racemosus is Mucor racemosus with the preservation number of CGMCC No.15264, the strain has high capability of producing linalool and linalyl acetate, and 9645.14 mu g/kg of linalool and 1228.05 mu g/kg of linalyl acetate can be produced after 90 days of post-fermentation, so that the prepared fermented bean curd has flower fragrance and fruit fragrance.
Biological material preservation information:
mucor racemosus (Mucor racemosus) is deposited in the China general microbiological culture Collection center on 2018, 01, 18 th month and the preservation number is as follows: CGMCC No.15264, preservation address: xilu No.1, Beijing, Chaoyang, Beijing, and institute for microbiology, China academy of sciences.
Drawings
In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings needed to be used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without creative efforts.
FIG. 1 shows Mucor racemosus (Mucor racemosus) CGMCC No.15264 and the colony morphology of Mucor racemosus on sour bean curd;
FIG. 2 shows the change of Mucor racemosus (Mucor racemosus) CGMCC No.15264 and the linalyl ester content of the Mucor racemosus for production in the fermentation process of the fermented bean curd;
FIG. 3 shows the change of Mucor racemosus (Mucor racemosus) CGMCC No.15264 and the linalyl acetate content of the Mucor racemosus for production in the fermentation process of the fermented bean curd;
FIG. 4 shows sensory fragrance evaluation results of Mucor racemosus (Mucor racemosus) CGMCC No.15264 and fermented bean curd produced by Mucor racemosus.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, embodiments of the present invention will be described in detail with reference to the accompanying drawings.
The first embodiment is as follows: mucor racemosus CGMCC No.15264 strain separation and identification method
(1) Obtaining appropriate dilution gradient and culturing
Weighing 25g of oil fermented bean curd obtained by making a natural fermentation in Chuxiong state of Yunnan province, adding the oil fermented bean curd into a homogenizing bag filled with 225mL of sterile water, and beating for 2min by using a beating type homogenizer; then sequentially diluting 1mL of bacterial liquid in 9mL of sterile water, and gradually diluting the sample homogeneous liquid (the liquid sample can comprise the stock solution) by 10 times to ensure that the concentration of the sample is diluted to 10 in a gradient manner-5Taking 4 dilutions to 102～105The sample homogenizing solution (liquid sample comprises stock solution) is prepared by respectively sucking 1mL of sample homogenizing solution into 2 sterile culture dishes at each dilution, pouring 20mL of potato-glucose-agar culture medium cooled to 46 ℃ into the culture dishes in time, rotating the culture dishes to mix the culture media uniformly, inverting the culture dishes after the agar is solidified, and culturing for 5 days at 28 +/-1 ℃.
(2) Separating and purifying
Typical single colonies were selected by dot-blot method, and the culture selection operation was repeated to obtain strains with excellent properties.
(3) Extraction of DNA
1) The strain on the medium was placed in a mortar beside the alcohol burner flame and ground with liquid nitrogen.
2) The ground bacteria were transferred to a 1.5mL centrifuge tube, the strain name was labeled, 0.6mL TE (pH 8.0) was added, and the mixture was pipetted uniformly to suspend the cells sufficiently.
3) Add 250. mu.L of 10% SDS, mix by gentle inversion.
4) Add 3. mu.L proteinase K (20 ng/. mu.L), mix gently, and water bath at 37 ℃ for 1 h.
5) Add 150. mu.L of 5mol/L NaCl and mix gently.
6) Add 150. mu.L of 2% CTAB, mix gently, and water bath at 65 ℃ for 20 min.
7) Centrifuging at 12000rpm for 20 min.
8) Carefully pipette the supernatant into a new 1.5mL centrifuge tube, add an equal volume of isopropanol, mix well, stand at room temperature for 30min, and centrifuge at 12000rpm for 10min at 4 ℃.
9) The supernatant was aspirated off, the liquid was dried on absorbent paper, 750. mu.L of 70% ethanol was added, the tube wall was flicked, the precipitate was suspended and inverted several times, and centrifuged at 12000rpm at 4 ℃ for 2 min.
10) Add 30. mu.L of purified water to each tube to dissolve the precipitate (RNase in water, final concentration 10 ng/. mu.L), flick the tube wall by hand and dissolve overnight at 4 ℃.
(4) Electrophoretic detection of DNA
The sample loading was 3. mu.L, and the Marker band consisted of, as measured on a 1% agarose gel at 100V: 100bp, 250bp, 500bp, 750bp, 1000bp, 2000bp, 3000bp and 5000 bp; the 750bp band concentration was 60 ng/3. mu.L.
(5) PCR amplification
18s primer sequence:
ITS1：5’-TCCGTAGGTGAACCTGCGG-3’(SEQ ID NO:1)；
ITS4：5’-TCCTCCGCTTATTGATATGC-3’(SEQ ID NO:2)；
the reaction system is shown in table 1, and the reaction conditions are shown in table 2:
table 1: reaction system:
table 2: reaction conditions are as follows:
(6) sequencing on the machine and outputting a peak diagram
(7) ITS sequence analysis and identification
According to a sequencing report given by Wuhan division of Huada Gene science and technology, the separated Mucor ITS sequence is compared and identified with the corresponding sequence of a known strain in a GenBank/EMBL/DDBJ database by combining with a BLAST analysis tool (http:// www.ncbi.nlm.nih.gov/BLAST), and is analyzed and identified as Mucor racemosus (Mucor racemosus) CGMCC No. 15264. The Mucor racemosus CGMCC No.15264 is a white large colony, the edge of the colony is villous, and the surface is dry; hypha morphological characteristics: hyphae have no rhizoid, hyphae have no diaphragm, cyst stalks do not form bundles, and the hyphae are single-grown, densely layered, vertical, single-axis branched and all terminal sporangia; the sporangia is large and ellipsoidal, and contains many spores; the spores are approximately round and 3-6 μm in size.
Example two: mucor racemosus CGMCC No.15264 fermented oil fermented bean curd
The mucor racemosus CGMCC No.15264 can be used for fermenting the oil fermented bean curd, can improve the content of linalool and linalyl acetate, and enhances the flower fragrance and fruit fragrance of the oil fermented bean curd.
1. Content change of linalool and linalyl acetate in fermentation process of fermented bean curd by mucor racemosus CGMCC No.15264
(1) GC-MS (gas chromatography-Mass Spectrometry) determination of linalool and linalyl acetate content in oil fermented bean curd fermentation process
The chromatographic conditions are as follows: an Rtx-WAX capillary column; column specification: 30m multiplied by 0.25 mu m multiplied by 0.25mm, the inlet temperature is 230 ℃, the column flow rate is 1mL/min, and the carrier gas is helium; temperature programming: the starting temperature was 45 deg.C (held for 1min), 5 deg.C/min to 185 deg.C (held for 1min), 10 deg.C/min to 240 deg.C (held for 8 min).
The mass spectrum conditions are as follows: the ionization mode EI has the electron energy of 70eV, the ion source temperature of 230 ℃, the interface temperature of 240 ℃ and the mass-to-charge ratio of 30-440.
(2) Fermentation of fermented bean curd
1) Preparation of a Mucor racemosus CGMCC No.15264 spore suspension: the Mucor racemosus CGMCC No.15264 stored at-80 deg.C is inoculated into PDA for cultivationCulturing in culture medium at 28 deg.C for 3 days, and subculturing for 3 times. Preparing mould spore into 105one/mL spore suspension (counted using a light microscope) was inoculated into liquid medium of PDA and cultured at 28 ℃ for 4 days; the spore suspension was diluted to 10 using sterile 0.85% normal saline5one/mL.
2) Preparing sour bean curd: selecting dry and plump fresh soybeans, removing impurities, and adding water in an amount which is 2-3 times of the volume of the soybeans for soaking for 12 hours; draining soaked soybeans, cleaning, adding clean water into the cleaned soybeans, grinding the cleaned soybeans with the soybean-water ratio of 1:8-1:10, grinding and filtering the ground soybeans, and collecting soybean milk; heating the raw soybean milk to boil and keeping for 5-10 min. Cooling soybean milk to about 58 deg.C, inoculating lactobacillus into the soybean milk, gelatinizing for 4 hr, stirring and crushing the prepared soybean curd, pouring into bean curd forming tank, and press-forming. Cutting bean curd into 3.5cm × 3.5cm × 2.0cm pieces.
3) Pre-fermentation of the oil fermented bean curd: will 105The Mucor racemosus CGMCC No.15264 spore suspension is sprayed onto bean curd and cultured at 25 deg.C and relative humidity of about 90% for 3 days.
4) Airing and salting: about 15% of water was removed.
5) After-fermentation of the fermented bean curd: uniformly mixing seasonings such as hot pepper, pepper and the like with the fermented bean curd blank, putting 12 blocks into a glass bottle, adding 100mL of rapeseed oil, and fermenting for 90 days.
Taking 2 bottles of samples respectively after primary fermentation for 0 day, primary fermentation for 4 days, airing, salting, secondary fermentation for 0 day, secondary fermentation for 30 days and secondary fermentation for 90 days, and measuring the content change of the volatile flavor substances.
(3) Mucor racemosus (Mucor racemosus) CGMCC No.15264 and colony morphology of Mucor racemosus fermented on sour bean curd for 4 days; the Mucor racemosus for production is purchased from China center for culture Collection of industrial microorganisms, and has the following strain preservation numbers: CICC 40491.
Mucor racemosus (Mucor racemosus) CGMCC No.15264 and colony morphology of Mucor racemosus fermented on sour bean curd for 4 days are shown in FIG. 1.
As shown in FIG. 1, after Mucor racemosus (Mucor racemosus) CGMCC No.15264 was inoculated to the sour bean curd for 4 days, the mycelia appeared white and dense; after the Mucor racemosus for production is inoculated on the sour bean curd for 4 days, the hyphae are mostly white, and the part is yellow and dense.
(4) Content change of linalool and linalyl acetate in fermented bean curd fermentation process
The content changes of linalool and linalyl acetate during the fermentation of the fermented bean curd are shown in fig. 2 and fig. 3, respectively. The content of linalool and linalyl acetate gradually increases with the extension of the fermentation time; after 90 days of post-fermentation, the linalool content in the fermented oil fermented bean curd of Mucor racemosus CGMCC No.15264 is 96454 mug/kg, and the content of the Mucor racemosus used for production is 3951 mug/kg; the content of linalyl acetate in the fermented oil fermented bean curd of Mucor racemosus CGMCC No.15264 is 1228 mu g/kg, while that of Mucor racemosus used for production is 322 mu g/kg.
2. Sensory evaluation of Mucor racemosus CGMCC No.15264 on aroma of fermented bean curd
Quantitative sensory evaluation of aroma was carried out on Mucor racemosus (Mucor racemosus) CGMCC No.15264 and fermented bean curd prepared from Mucor racemosus, and the sensory evaluation criteria of aroma are shown in Table 3, and the results are shown in FIG. 4.
TABLE 3 Scent criteria for fragrance
The experimental result shows that the oil fermented bean curd prepared by the fermentation of CGMCC No.15264 has obvious salty, oil and fruit flavors; the production process of the oil fermented bean curd is pure bacteria fermentation, so that the controllability is remarkable, the possibility of over fermentation is reduced, and the mould taste of the final product is not remarkable.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and simplifications made in the spirit of the present invention are intended to be included in the scope of the present invention.
SEQUENCE LISTING
<110> university of south of the Yangtze river
<120> preparation method of oil fermented bean curd with high fruit aroma and flower aroma yield
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<170> PatentIn version 3.3
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taaggataga cattggaaat gttaaccgag tcataatcag gtttaggcct ggtatcctat 180
tattatttac caaatgaatt cagaattaat attgtaacat agacctaaaa aatctataaa 240
acaactttta acaacggatc tcttggttct cgcatcgatg aagaacgtag caaagtgcga 300
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ttggtattcc aatgagcacg cctgtttcag tatcaaaaca aaccctctat ccaacttttg 420
ttgtatagga ttattggggg cctctcgatc tgtatagatc ttgaaatccc tgaaatttac 480
taaggcctga acttgtttaa atgcctgaac ttttttttaa tataaaggaa agctcttgta 540
attgactttg atggggcctc ccaaataaat cttttttaaa tttgatctga aatcaggcgg 600
gattacccgc tgaacttaag cata 624
Claims (7)
1. A preparation method of oil fermented bean curd with high flower fragrance and fruit fragrance is characterized in that sour bean curd is used as a raw material, Mucor racemosus (Mucor racemosus) spore suspension is sprayed on the surface of the sour bean curd for pre-fermentation, and then airing and salting are carried out, and seasoning is added for post-fermentation; wherein the Mucor racemosus is preserved in the China general microbiological culture Collection center in 2018, month 01 and day 18, and the preservation numbers are as follows: CGMCC No.15264, the preservation address is No. 3 of the institute of microbiology of the national academy of sciences of China, institute of Xilu No.1 of Beijing, Chaoyang.
2. The method according to claim 1, wherein the sour bean curd is obtained by inoculating lactic acid bacteria into soybean milk and gelling for 3-5 hours; the preparation steps are as follows: stirring and crushing the prepared soybean curd, then pouring the soybean curd into a bean curd forming groove, and pressing and forming.
3. The method of claim 1, wherein the concentration of the Mucor racemosus spore suspension is 105one/mL.
4. The method as claimed in claim 1, wherein the airing time is 10-14 hours, and the salting parameter is adding 5% salt for 12-14 hours, and removing 12-18% of water.
5. The method as claimed in claim 1, wherein the seasoning is added for post-fermentation by mixing the seasoning and the fermented bean curd base uniformly, taking 12 pieces and putting into a glass bottle, adding 100mL rapeseed oil, and performing post-fermentation for 90 days.
6. The method of claim 5, wherein the seasoning comprises pepper and zanthoxylum.
7. The method of claim 2, further comprising, after said press forming: the bean curd was cut into 3.5cm by 2.0 cm.
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| CN101658273A (en) * | 2009-09-17 | 2010-03-03 | 北京王致和食品集团有限公司 | Method for producing low-salt fermented bean curd |
| CN106591280A (en) * | 2016-12-13 | 2017-04-26 | 惠州市冠益食品有限公司 | Mutation screening method of Mucor sufu strain, fermented bean curd, and production method of fermented bean curd |
| CN107629968A (en) * | 2017-10-31 | 2018-01-26 | 云南羊泉生物科技股份有限公司 | A kind of purification of fermented bean curd strain and its application process |
| CN109082384A (en) * | 2018-08-20 | 2018-12-25 | 江南大学 | Mucor racemosus and its application of a kind of high yield linalool and bergamio |
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| PL2774481T3 (en) * | 2013-03-08 | 2019-03-29 | Symrise Ag | Antimicrobial compositions |
| CN104087511A (en) * | 2014-04-11 | 2014-10-08 | 湖南农业大学 | Mucor racemosus strain and its application |
| EP3085239A1 (en) * | 2015-04-25 | 2016-10-26 | DMK Deutsches Milchkontor GmbH | Sterile cheese base |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101658273A (en) * | 2009-09-17 | 2010-03-03 | 北京王致和食品集团有限公司 | Method for producing low-salt fermented bean curd |
| CN106591280A (en) * | 2016-12-13 | 2017-04-26 | 惠州市冠益食品有限公司 | Mutation screening method of Mucor sufu strain, fermented bean curd, and production method of fermented bean curd |
| CN107629968A (en) * | 2017-10-31 | 2018-01-26 | 云南羊泉生物科技股份有限公司 | A kind of purification of fermented bean curd strain and its application process |
| CN109082384A (en) * | 2018-08-20 | 2018-12-25 | 江南大学 | Mucor racemosus and its application of a kind of high yield linalool and bergamio |
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