CN113755398A - Dairy fan fermentation strain with aroma enhancement function, dairy fan leavening agent and application thereof - Google Patents
Dairy fan fermentation strain with aroma enhancement function, dairy fan leavening agent and application thereof Download PDFInfo
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- CN113755398A CN113755398A CN202111180264.6A CN202111180264A CN113755398A CN 113755398 A CN113755398 A CN 113755398A CN 202111180264 A CN202111180264 A CN 202111180264A CN 113755398 A CN113755398 A CN 113755398A
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Images
Classifications
-
- 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
- A23C9/00—Milk preparations; Milk powder or milk powder preparations
- A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
- A23C9/127—Fermented milk preparations; Treatment using microorganisms or enzymes using microorganisms of the genus lactobacteriaceae and other microorganisms or enzymes, e.g. kefir, koumiss
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/11—Lactobacillus
- A23V2400/169—Plantarum
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- Life Sciences & Earth Sciences (AREA)
- Microbiology (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Dairy Products (AREA)
Abstract
The invention discloses a milk fan fermentation strain with an aroma enhancement function, a milk fan leavening agent and application thereof. The artificial milk fan fermentation strain of the invention is lactobacillus plantarum ML9 and rhodotorula mucilaginosa 2-3. The two strains generate different types and contents of aroma compounds, and the aroma generation performance is complementary. The lactobacillus plantarum ML9 and the rhodotorula mucilaginosa 2-3 are added into the traditional acid water for fermentation to obtain the milk fan starter, the milk fan starter is compared with the milk fan fermented by the traditional acid water, the types, the content proportion and the artificial sensory evaluation result of key aroma components are taken as main indexes, and the milk fan starter after strain compounding can achieve the purposes of complementary aroma production advantages and optimized flavor. The dairy fan prepared by the dairy fan leavening agent has harmonious overall aroma, good and stable sensory properties and strong practicability, and the dairy fan product produced by the dairy fan leavening agent has better flavor and improves the overall flavor quality of the dairy fan.
Description
Technical Field
The invention relates to a milk fan fermentation strain with an aroma enhancement function, a milk fan leavening agent and application thereof, and belongs to the technical field of biological fermentation.
Background
The milk fan is a special fermented milk product in Yunnan area of China, and belongs to stretched type yoghurt. The cheese is rich in nutrition and unique in flavor, is popular with consumers in southwest areas of China, and is beneficial to filling up the gap of market share of domestic cheese.
At present, a plurality of problems still exist in the production of the dairy fan, and the main reason is that the production of the dairy fan still continues to be in the traditional household manual workshop type. The lagging of the production process and equipment causes the low yield and the insufficient yield compared with the industrial production; meanwhile, most dairy fans are manufactured by manufacturers according to self experiences, no standard can be used for reference, and the produced products have different qualities such as flavor and the like. The sensory quality of cheese products is an important factor affecting their consumer acceptance, with the flavor profile being the most immediate and important sensory quality directly affecting the consumer's buying intent. The flavor of the milk fan is mainly given by volatile compounds in the milk fan, and the compounds are mainly generated in microbial metabolic reactions such as fat, protein hydrolysis and lactose metabolism in the milk fan. The problems of unstable flavor quality, unclear flavor substance forming mechanism and the like of the dairy fan are just the bottleneck of the standardized production of products, and the industrial production process of the dairy fan is severely limited.
The activity of microorganisms is the key of flavor formation in the cheese maturation process, and the microbial flora succession rule and the metabolic change of flavor substances have certain relevance in the cheese maturation process. In recent years, a number of studies have shown that the addition of specific strains can increase the concentration of certain specific aroma compounds and thus improve the flavor of cheese. In the case of starter, sour water, of dairy fan, the initial microbial composition can greatly affect the microbial composition in the dairy fan product, which in turn has an effect on the flavor of the dairy fan. However, it is difficult for a single strain to achieve a good aroma improving effect, and it also affects the production of a dairy fan.
Disclosure of Invention
The technical problem to be solved by the invention is as follows: aiming at the problems of inconsistent microbial composition and unstable flavor characteristics caused by the absence of a fixed leaven in the conventional handmade cheese milk fan.
In order to solve the technical problems, the invention provides a milk fan fermentation strain with a fragrance-enhancing function, which is at least one of lactobacillus plantarum ML9 and rhodotorula mucilaginosa 2-3;
the lactobacillus plantarum ML9 has a latin name of Lactplantibacillus plantarum ML9 and a preservation number of CCTCC M2021745, is preserved in China center for type culture Collection, and is preserved in Wuchang district, Wuhan City, Hubei province in eight Lou of Wuchang, and the preservation date is 2021 year, 6 months and 23 days.
The Latin name of the Rhodotorula mucilaginosa 2-3 is Rhodotorula mucor 2-3, the preservation number is CCTCC M2021747, the preservation unit is China center for type culture Collection, the preservation address is eight Lopa Alojia mountain in Wuchang district, Wuhan City, Hubei province, and the preservation date is 2021 year, 6 months and 23 days.
The invention also provides application of the milk fan fermentation strain with the aroma enhancement function in preparation of a milk fan leavening agent.
The invention also provides a milk fan leavening agent with the flavor enhancing function, which is acid water fermented by the milk fan leavening strain with the flavor enhancing function in claim 1.
Preferably, the milk fan starter comprises acid water A fermented by lactobacillus plantarum ML9 and acid water B fermented by rhodotorula mucilaginosa 2-3.
Preferably, the volume ratio of the acid water A to the acid water B is 1: 1.
Preferably, the preparation method of the milk fan starter comprises the following steps:
step 1: preheating raw milk, adding traditional acid water, continuously heating until the milk is solidified;
step 2: filtering out the coagulated block by using a filter screen, and obtaining new making acid water by remaining whey;
and step 3: preparing acid water, inoculating lactobacillus plantarum ML9, and fermenting at room temperature to obtain acid water A;
and 4, step 4: preparing fresh acid water, inoculating Rhodotorula mucilaginosa 2-3, and fermenting at room temperature to obtain acid water B;
and 5: mixing the acid water A and the acid water B to obtain the dairy fan starter.
Preferably, the temperature of preheating and continuous heating in the step 1 of the preparation method is 65 ℃; the mass ratio of the raw milk to the traditional acid water is 1: 1.
Preferably, the inoculation concentration of the lactobacillus plantarum ML9 in the step 3 of the preparation method is 2.0-2.8 x 104CFU/mL, and the fermentation time is 5-6 days.
Preferably, the inoculation concentration of the rhodotorula mucilaginosa 2-3 in the step 4 of the preparation method is 5.2-6.0 x 105CFU/mL, and the fermentation time is 5-6 days.
The invention also provides application of the milk fan leavening agent with the fragrance enhancing function in preparing milk fans.
Compared with the prior art, the invention has the beneficial effects that:
1. according to the invention, the artificial dairy fan starter with the aroma-increasing function is obtained by selecting the appropriate aroma-producing strains and determining the proportion of the core aroma-producing strains, so that the problem of unstable batch-to-batch flavor characteristics caused by the absence of a fixed starter in the conventional dairy fan production is effectively solved;
2. the artificial milk fan starter with the aroma enhancement function, provided by the invention, comprises a plurality of bacterial strains which completely use the milk fan as a source and play an important role in the flavor of the milk fan, so that the prepared milk fan is superior to the milk fan prepared by the traditional acid water in the flavor characteristic, and the defect of the traditional acid water preparation of the milk fan in the flavor is made up;
3. compared with the fragrance profile of the dairy fan prepared from the traditional acid water by adopting the technical means of the electronic nose, the fragrance profile of the dairy fan prepared from the acid water fermented by the artificial dairy fan leavening agent is found to be improved in the content of various fragrance substances and the overall fragrance intensity, and the artificial dairy fan leavening agent can effectively enhance the overall fragrance of the dairy fan.
Drawings
FIG. 1 shows the correlation between the core flora of milk fans and the volatile aroma component (OAV ≧ 1);
FIG. 2 shows the correlation analysis of microorganisms with volatile compounds during the manufacturing process of a milk fan;
fig. 3 shows an electronic nasal aroma profile analysis of two breast fans.
Deposit description
Lactobacillus plantarum ML9 (Lactplantibacillus plantarum ML9) is preserved in the China center for type culture Collection, the eight Lopa Lobata mountain in Wuchang district, Wuhan City, Hubei province, with the preservation date of 2021 year, 6 months and 23 days and the preservation number of CCTCC M2021745.
Rhodotorula mucilaginosa 2-3(Rhodotorula mucilaginosa 2-3) is preserved in the eight Lopa in Wuchang district of Wuhan city, Hubei province in China, with a preservation date of 2021 year, 6 months and 23 days and a preservation number of CCTCC M2021747.
Detailed Description
In order to make the invention more comprehensible, preferred embodiments are described in detail below with reference to the accompanying drawings.
The following methods were used to identify the species in the examples below:
16S rDNA/ITS sequence identification is carried out on the strain. Before identification, the DNA sequence is subjected to PCR amplification, and the reaction system is as follows: 2 μ L of NL1 (5'-GCATATCAATAAGCGGAGGAAAAG-3') (SEQ ID NO: 1); 2 μ L of NL4 (5'-GGTCCGTGTTTCAAGACGG-3') (SEQ ID NO: 2); 2. mu.L of DNA; 19 μ L of ddH2O; 25 μ L of 2 Xgold Star Best Mixed. The PCR reaction program is: (1)95 ℃ for 10min, (2)94 ℃ for 30S, (3)55 ℃ for 30S, (4)72 ℃ for 20S, repeating the steps (2) to (4) for 30 cycles, (5)72 ℃ for 5min, and (6) storing at 4 ℃.2 μ L of 27F primer (5'-GAGAGTTTGATCCTGGCTCAG-3') (SEQ ID NO: 3); 2 μ L of 1492r primer (5'-TACGGCTACCTTGTTACGAC-3') (SEQ ID NO: 4); 2. mu.L of DNA; 19 μ L of ddH2O; 25 μ L of 2 Xgold Star Best Mixed. The PCR reaction program is: (1) 10min at 95 deg.C, (2) 30s at 94 deg.C, (3) 30s at 55 deg.C, (4) 1.5min at 72 deg.C, repeating the steps (2) - (4) for 30 cycles, (5) 5min at 72 deg.C, and (6) storing at 4 deg.C. And then carrying out gel electrophoresis detection on the PCR product by using 1.0% agarose, carrying out electrophoresis in 0.5 × TAE electrophoresis solution for 20min under the voltage of 100V, and then detecting the extraction effect on a gel imager, wherein if a clear band is visible and no obvious nonspecific amplification exists, the success of PCR can be judged. Then the 16S rDNA sequence of the strain is compared with the known strain sequence in GenBank.
The conventional acid water used in the following examples was prepared by the following method: adding water into immature fresh green papaya or dry papaya, boiling, decocting with slow fire for half an hour, and filtering to obtain acid water.
Example 1
Analysis of core flora in milk fans:
conveying milk fan samples from six areas (Kunming, Yuxi, Dengchang, Xizhou, Jianchuan and Wei mountain) in Yunnan to a laboratory at low temperature, mashing the samples by using a sterile mortar, weighing 10g of the ground samples into powder, putting the powder into 90mL of sterile physiological saline (0.85g/100mL), and uniformly mixing to obtain a suspension for later use. And centrifuging the uniformly mixed suspension for 2min at the speed of 5000r/min, collecting the centrifuged thalli and placing the thalli in an ice box. Collected thallus is subjected to fecal genome DNA extraction by utilizing TIANAmp Stool DNA KitDNA extraction was performed. The extracted DNA was stored at-20 ℃. The extracted DNAs were subjected to PCR reaction using primers NL1 (5'-GCATATCAATAAGCGGAGGAAAAG-3') (SEQ ID NO:1), NL4 (5'-GGTCCGTGTTTCAAGACGG-3') (SEQ ID NO:2) and primers 27F (5'-GAGAGTTTGATCCTGGCTCAG-3') (SEQ ID NO:3), 1492r (5'-TACGGCTACCTTGTTACGAC-3') (SEQ ID NO:4) in a 50.0. mu.L PCR reaction system containing 2. mu.L of DNA, 25. mu.L of 2 XGoldStar Best MasterMix, 2. mu.L of forward primer (100. mu.M), 2. mu.L of reverse primer (100. mu.M), and ddH using ddH2O make up to 50.0. mu.L. The PCR reaction program was (primers NL1 and NL 4): (1)95 ℃ for 10min, (2)94 ℃ for 30s, (3)55 ℃ for 30s, (4)72 ℃ for 20s (1.5 min when primers 27F and 1492 r), repeating the steps (2) - (4) for 30 cycles, (5)72 ℃ for 5min, and (6) storing at 4 ℃. And then carrying out gel electrophoresis detection on the PCR product by using 1.0% agarose, carrying out electrophoresis in 0.5 × TAE electrophoresis solution for 20min under the voltage of 100V, and then detecting the extraction effect on a gel imager, wherein if a clear band is visible and no obvious nonspecific amplification exists, the success of PCR can be judged. PCR amplification of the target fragment was performed using bacterial 16SrDNA primers 338F (5'-ACTCCTACGGGAGGCAGCAG-3') (SEQ ID NO:5) and 806R (5 '-GGACTACHVGGGTWTCTAAT-3') (SEQ ID NO:6), fungal ITS primers ITS1F (5'-CTTGGTCATTTAGAGGAAGTAA-3') (SEQ ID NO:7) and ITS2R (5'-GCTGCGTTCATCGATGC-3') (SEQ ID NO: 8). The PCR reaction system was 20.0. mu.L (primers 338F and 806R) including 2. mu.L of 2.5mM dNTPs, 4. mu.L of 5 XFastPfu Buffer (2. mu.L of 10 Xbuffer in the case of primers ITS1F and ITS2R), 0.8. mu.L of Forward Primer (5. mu.M), 0.8. mu.L of Reverse Primer (5. mu.M), 0.4. mu.L of FastPfu Polymerase (0.2. mu.L of rTaq Polymerase in the case of primers ITS1F and ITS2R), 0.2. mu.L of BSA, 10ng Template DNA, using ddH2O make up to 20.0. mu.L. The PCR reaction program was (primers 338F and 806R, ITS1F and ITS 2R): (1)95 ℃ for 3min, (2)95 ℃ for 30s, (3)55 ℃ for 30s, (94)72 ℃ for 45s, and the steps (2) to (4) are repeated for 35 cycles, (5)72 ℃ for 10min, and (6)10 ℃ until stop. And (3) recovering the product by using glue, and performing fluorescence quantification on the PCR amplification recovered product according to the preliminary quantification result of electrophoresis, wherein the fluorescence reagent is Quant-iT PicoGreen dsDNA Assay Kit, and the quantification instrument is a Microplate reader (BioTek, FLx 800). Sequencing the amount of each sample based on the quantitative results of fluorescenceAnd (3) mixing the samples according to the corresponding proportion as required. Finally, metagenomic sequencing is carried out by using a Miseq platform.
The microorganisms from milk fan samples from six regions (Kunming, Yuxi, Dengchuan, Happy continent, Jianchuan and Wei mountain) were sequenced by high throughput sequencing techniques. The dominant bacteria found in six regional milk fan samples with abundance content of more than 1% were Lactococcus (Lactococcus, 39.96%), Lactobacillus (Lactobacillus, 16.65%), ralstonia (Raoultella, 8.38%), Streptococcus (Streptococcus, 7.32%), Acetobacter (Acetobacter, 4.99%), Acinetobacter (acietobacter, 3.44%), Klebsiella (Klebsiella, 2.54%), Leuconostoc (Leuconostoc, 2.44%), chrysotobacter (Chryseobacterium, 2.16%), aquabacter (enterobacter, 1.59%), Enterococcus (Enterococcus, 1.45%) and Pseudomonas (Pseudomonas, 1.28%), respectively; the dominant fungi with abundance content of more than 1% are Rhodotorula (Rhodotorula, 36.09%), torula sporogenes (torula sporora, 17, 65%), Candida (Candida, 16.61%), ledebur (Lodderomyces, 7.82%), saccharomyces carlsbergii (Naganishia, 5.83%), (cytofilobinium, 2.98%), Trichosporon (Trichosporon, 2.94%), Pichia (Pichia, 2.85%), debkkera (Dekkera, 2.55%) and Kluyveromyces (Kluyveromyces, 2.52%), respectively.
Example 2
Correlation analysis of the core bacteria group and the volatile aroma component (OAV is more than or equal to 1) in the milk fan:
SPME-GC-MS was used to determine volatile aroma compounds in samples from six regional milk fans. The specific method comprises the following steps: accurately weighing 4.0g of fermentation sample in a 15mL extraction bottle, adding 100 μ L of 20 mg/L2-octanol serving as an internal standard substance, balancing for 5min in a constant-temperature water bath kettle at 60 ℃, inserting an extraction head device (75 μm CAR/PDMS) aged for 20min into the 15mL extraction bottle, and extracting for 40min at 60 ℃.
Then, after the extraction is finished, the extraction head is pushed into a gas phase injection port at 250 ℃ for desorption for 5 minutes, and the extracted volatile compounds are analyzed and identified by combining GC-MS. The gas chromatography conditions were as follows: a chromatographic column: HP-INNOWAX (60 m.times.0.25 mm.times.0.25 μm); sample inlet temperature: 250 ℃; temperature programming: maintaining at 40 deg.C for 4min, increasing the temperature to 100 deg.C at 4 deg.C/min, maintaining for 2min, increasing the temperature to 150 deg.C at 3 deg.C/min, increasing the temperature to 230 deg.C at 10 deg.C/min, and maintaining for 5 min; carrier gas: helium (99.99% pure); flow rate: 1 mL/min; and (3) sample introduction mode: no split-flow sample introduction. Mass spectrum conditions: electron bombardment energy: 70 eV; ion source temperature: 230 ℃; temperature of the quadrupole rods: 150 ℃; emission current: 35 muA; scanning speed: 1.9 scans/s; mass scan range: 30-450 amu. After each desorption, the extraction head was aged in an aging apparatus at 250 ℃ for 20 min. Each sample was analyzed in duplicate 3 times.
Finally, 13 compounds (OAV is more than or equal to 1) contributing to the aroma are qualitatively and quantitatively analyzed, and the compounds comprise 7 acids, 4 esters, 1 aldehyde and 1 phenolic substance. Pearson correlation coefficients between 13 main volatile components in the milk fan sample and the core flora are calculated and visualized by cytoscape software, and the result is shown in FIG. 1. The results show that there is a significant positive correlation between lactobacillus and caprylic acid, caproic acid, butyric acid, ethyl caproate and ethyl caprylate, respectively. The dominant fungus genus Rhodotorula has obvious positive correlation with heptanoic acid, octanoic acid, decanoic acid and isobutyl butyrate respectively. The two bacteria have great contribution to the formation of the flavor of the dairy fan.
Example 3
Screening and verifying the dominant bacteria producing fragrance in milk fan and acid water:
analyzing the correlation between volatile compounds changed in the manufacturing process of the milk fan and dominant bacteria, respectively adding 10 strains of lactobacillus and 7 strains of rhodotorula obtained by morphological and 16S rRNA/ITS gene sequence analysis and identification into the traditional acid water fermentation for manufacturing the milk fan for analysis, and screening the optimal flavor-producing dominant bacteria for improving the overall flavor of the milk fan in the milk fan leavening agent.
The method for measuring the change of the volatile compounds in the manufacturing process of the milk fan comprises the following specific steps:
1) taking fresh sour water and milk fan samples collected from Eryuan county of Dalizhou, Yunnan province as raw materials, sampling on days 0, 3 and 6 after acid water fermentation, and collecting five samples marked as S0(0-1, 0-2, 0-3, 0-4, 0-5), S3(3-1, 3-2, 3-3, 3-4, 3-5) and S6(6-1, 6-2, 6-3, 6-4, 6-5) at each time point, wherein the acid water samples are fully stirred and then sampled from the upper part, the middle part and the bottom part of a fermentation tank respectively; samples were taken at 0 and 24 hours after the completion of the production of the fan, 5 samples, designated R0(0-1, 0-2, 0-3, 0-4, 0-5) and R24(24-1, 24-2, 24-3, 24-4, 24-5), were taken at each time point, and one whole fan was taken for each sample. The collected sample is sealed in a low-temperature vacuum manner;
2) headspace solid phase microextraction: a3 g sample of a dairy fan was weighed into a 15mL extraction flask and 100. mu.L of 2-octanol (13. mu.g/L) was added as an internal standard. The extraction flask is equilibrated in a 60 deg.C constant temperature water bath for 5min, and the extraction head device aged for 20min is inserted therein, and extracted at 60 deg.C for 35 min. After extraction is finished, inserting an extraction head into an aged gas chromatography-mass spectrometer for detection and analysis of volatile compounds;
3) GC-MS analysis of samples for volatile compounds:
chromatographic conditions are as follows: a chromatographic column: HP-INNOWAX (60 m.times.0.25 mm.times.0.25 μm); sample inlet temperature: 250 ℃; temperature programming: keeping at 40 deg.C for 4min, increasing to 100 deg.C at 3 deg.C/min, keeping for 2min, increasing to 150 deg.C at 4 deg.C/min, increasing to 230 deg.C at 10 deg.C/min, keeping for 5 min: carrier gas: helium (99.99% pure); flow rate: 1 mL/min; and (3) sample introduction mode: no shunt sampling;
mass spectrum conditions: an ion source: electron ionization; ionization energy: 70 eV; ion source temperature: 230 ℃; temperature of the quadrupole rods: 150 ℃; emission current: 35 muA; scanning speed: 1.9 scans/s; mass scan range: 50-550 amu.
The measurement is repeated 3 times for each sample, and as shown in table 1, 16 compounds such as ethyl acetate are screened by calculating OAV value after the compounds are qualitatively and quantitatively determined. The relevance of dominant bacteria and 16 volatile compounds in the dairy fan and acid water is analyzed, as shown in figure 2, strong relevance exists between aldehydes substances which are obviously related to the mature flavor of the dairy fan in lactobacillus and rhodotorula, and the contribution of the lactobacillus and the rhodotorula to the flavor of the dairy fan is large.
TABLE 1 changes in volatile Compounds during the manufacturing Process of the milk Fan
RI in Table 1 represents the retention index of volatile compounds on an HP-Innowax column; a indicates that the calculated value of retention index is consistent with the literature value; b represents an identification method: MS, mass spectral comparison using nist11.l database; nd indicates that it was not detected in the sample.
The method for verifying the aroma-producing capability of the aroma-producing bacteria comprises the following specific steps:
1) respectively activating the lactobacillus plantarum strain and the rhodotorula rubra strain obtained by identification, inoculating the activated lactobacillus plantarum strain and the rhodotorula rubra strain into newly prepared acid water, and fermenting for 6 days to obtain mature acid water;
2) adding acid water and traditional acid water to be respectively fermented by the aroma-producing bacteria to be verified 1:1 mixing acid water for making milk fan;
3) applying acid water prepared by adding aroma-producing bacteria for fermentation and compounding with traditional acid water to the manufacture of the milk fan according to the milk fan manufacturing method;
4) analysis of milk fan flavor: the method for analyzing the flavor of the dairy fan is the same as the method for measuring the volatile aroma compounds in the dairy fan sample by using SPME-GC-MS in example 2;
5) the best aroma-producing dominant bacteria among the 17 aroma-contributing bacteria are screened by taking volatile compounds changed in the manufacturing process of the dairy fan and compounds related to microorganisms in the dairy fan as indexes.
As shown in Table 2, the types of the corresponding volatile compounds detected in the milk fans added with Lactobacillus plantarum-2, Lactobacillus plantarum-4 and Lactobacillus casei-1 are more, and the 3 strains of the milk fans produced by Lactobacillus plantarum-4 are significantly higher (p <0.05) than other strains, such as ethyl caprylate, butyric acid, hexanal and nonanal; as shown in Table 3, in the milk fans to which the Rhodotorula mucilaginosa-3 and Rhodotorula mucilaginosa-4 were added, all the relevant volatile compounds were detected, and the concentration of various substances in the milk fan to which the Rhodotorula mucilaginosa-3 was added was significantly (p <0.05) higher than that of the milk fan to which the Rhodotorula mucilaginosa-4 was added. Therefore, Lactobacillus plantarum-4 and Rhodotorula mucilaginosa-3 are the best flavor-producing dominant bacteria. 16S rDNA/ITS sequence identification is carried out on the two strains, and the two strains are compared with the known strain sequences in GenBank, the lactobacillus plantarum-4 is lactobacillus plantarum ML9, the latin name of the lactobacillus plantarum ML9 is Lactplantibibacillus plantarum, and the preservation number is CCTCC M2021745; the Rhodotorula mucilaginosa-3 is Rhodotorula mucilaginosa 2-3, and its Latin name is Rhodotorula mucina 2-3, and its preservation number is CCTCC M2021747.
TABLE 2 Lactobacillus screening results
In table 2, nd indicates that the substance was not detected in the sample, and different superscript letters indicate significant differences (p < 0.05).
TABLE 3 Rhodotorula screening results
In table 3, nd indicates that the substance was not detected in the sample, and different superscript letters indicate significant differences (p < 0.05).
Example 4
Preparing a leavening agent of the artificial dairy fan and preparing the dairy fan:
a milk fan leaven with flavor enhancement function consists of (according to inoculation concentration): lactobacillus plantarum 2.42X 104CFU/mL. The inoculation concentration is determined by the amount of acid water added during the curd process, the total number of bacterial and fungal colonies in the milk fan, and the abundance of the Corynebacteria level in the milk fan.
The acid water preparation method comprises the following specific steps:
1) preheating: heating raw milk to 65 ℃;
2) curding: according to the mass ratio of 1:1, adding traditional acid water into the milk, continuously heating until the milk is solidified;
3) and (3) filtering: filtering out the coagulated block by using a filter screen, and obtaining new making acid water by remaining whey;
4) fermentation: and (4) collecting the acid water prepared in the step (3) in a container, inoculating lactobacillus plantarum strain, and fermenting at room temperature for 5-6 days to obtain the acid water.
The preparation method of the milk fan comprises the following specific steps:
1) preheating: heating raw milk to 65 ℃;
2) curding: according to the mass ratio of 1:1, adding the acid water into the milk, and continuously heating until the milk is solidified;
3) blanching: adjusting the temperature to 50 ℃, filtering off whey by using a filter screen, and kneading the curd into blocks by using a clamp;
4) and (3) stretching and forming: taking out the condensed milk block, stretching the condensed milk block into a fan shape, and winding the fan-shaped condensed milk block on an airing rod;
5) air drying: hanging the drying rod outdoors, and naturally drying for 24h to obtain the milk fan.
Example 5
Preparing a leavening agent of the artificial dairy fan and preparing the dairy fan:
a milk fan leaven with flavor enhancement function consists of (according to inoculation concentration): rhodotorula mucilaginosa 5.60X 105CFU/mL. The inoculation concentration is determined by the amount of acid water added during the curd process, the total number of bacterial and fungal colonies in the milk fan, and the abundance of the Corynebacteria level in the milk fan. The acid water preparation method comprises the following specific steps:
1) preheating: heating raw milk to 65 ℃;
2) curding: according to the mass ratio of 1:1, adding traditional acid water into the milk, continuously heating until the milk is solidified;
3) and (3) filtering: filtering out the coagulated block by using a filter screen, and obtaining new making acid water by remaining whey;
4) fermentation: and (4) collecting the acid water prepared in the step (3) in a container, adding the rhodotorula mucilaginosa strain, and fermenting for 5-6 days at room temperature to prepare the acid water.
The preparation method of the milk fan comprises the following specific steps:
1) preheating: heating raw milk to 65 ℃;
2) curding: according to the mass ratio of 1:1, adding the acid water into the milk, and continuously heating until the milk is solidified;
3) blanching: adjusting the temperature to 50 ℃, filtering off whey by using a filter screen, and kneading the curd into blocks by using a clamp;
4) and (3) stretching and forming: taking out the condensed milk block, stretching the condensed milk block into a fan shape, and winding the fan-shaped condensed milk block on an airing rod;
5) air drying: hanging the drying rod outdoors, and naturally drying for 24h to obtain the milk fan.
Example 6
Preparing a leavening agent of the artificial dairy fan and preparing the dairy fan:
the acid water obtained in examples 4 and 5 was mixed in a volume ratio of 1:1 to obtain the artificial milk fan starter.
The preparation method of the milk fan comprises the following specific steps:
1) preheating: heating raw milk to 65 ℃;
2) curding: according to the mass ratio of 1:1, adding the acid water starter into cow milk, and continuously heating until the cow milk is solidified;
3) blanching: adjusting the temperature to 50 ℃, filtering off whey by using a filter screen, and kneading the curd into blocks by using a clamp;
4) and (3) stretching and forming: taking out the condensed milk block, stretching the condensed milk block into a fan shape, and winding the fan-shaped condensed milk block on an airing rod;
5) air drying: hanging the drying rod outdoors, and naturally drying for 24h to obtain the milk fan.
Comparative example 1
Traditional sour water preparation milk fan:
1) preheating: heating raw milk to 65 ℃;
2) curding: according to the mass ratio of 1:1, adding traditional acid water into the milk, continuously heating until the milk is solidified;
3) blanching: adjusting the temperature to 50 ℃, filtering off whey by using a filter screen, and kneading the curd into blocks by using a clamp;
4) and (3) stretching and forming: taking out the condensed milk block, stretching the condensed milk block into a fan shape, and winding the fan-shaped condensed milk block on an airing rod;
5) air drying: hanging the drying rod outdoors, and naturally drying for 24h to obtain the milk fan.
Effect example 1
Respectively adding lactobacillus plantarum and rhodotorula mucilaginosa to ferment the volatile compounds of the milk fan, and comparing the types:
the volatile aroma components of the milk fan were determined by SPME-GC-MS. The specific method comprises the following steps: firstly, solid-phase microextraction is carried out, 3g of milk fan samples are placed in a 15mL headspace bottle, 100 muL of internal standard 2-octanol (13 mug/L) is added, the headspace bottle is placed in a 60 ℃ water bath kettle, balance is carried out for 5min, and then an SPME extraction head aged for 20min is inserted into the headspace bottle to extract the milk fan samples for 35 min. GC conditions, column HP-INNOWAX (60 m.times.0.25 mm. times.0.25 μm); the temperature of a sample inlet is 250 ℃; a temperature raising program, wherein the initial temperature is kept at 40 ℃ for 4min, the temperature is raised to 100 ℃ at 3 ℃/min, then kept for 2min, then raised to 150 ℃ at 4 ℃/min, and finally raised to 230 ℃ at 10 ℃/min, and kept for 5 min; the carrier gas is helium (with the purity of 99.99 percent) and the flow rate is 1 mL/min; the sample introduction mode is non-shunting sample introduction. MS conditions, electron ionization ion source; electron energy 70 eV; the ion source temperature is 230 ℃; temperature of the quadrupole rods: 150 ℃; emission current: 35 muA; scanning speed: 1.9 scans/s; the mass scanning range is 50-550 amu.
The volatile fragrance types of examples 4 and 5 were compared with those of comparative example 1, as shown in Table 4.
TABLE 4 GC-MS identification results of fermented milk fans of different strains
As can be seen from Table 4, the contents of acetic acid, butyric acid, caproic acid, capric acid, ethanol and pentanol in the milk fan made of the acid water to which Rhodotorula mucilaginosa 2-3 (preservation number: CCTCC M2021747) was added were increased. The content of esters such as ethyl acetate, isoamyl acetate, ethyl hexanoate and ethyl octanoate in the milk fan made of acid water added with Lactobacillus plantarum ML9 (CCTCC M2021745) is increased.
Effect example 2
The fragrance profile of the milk fan prepared by the artificial milk fan leaven is compared with that of the milk fan prepared by the traditional acid water:
the milk fan aroma was analyzed using an electronic Nose (Supernose, Isonso Group Corporation, New York, USA) with 14 sensors and a Smart Nose Intelligent recognition software system. Accurately weighing 15.0g of each milk fan sample in a sample bottle, standing for 30min, carrying out electronic nose analysis, manually injecting a single sample by adopting a headspace aspiration method, directly inserting a sample injection needle into the sample bottle, and carrying out zero clearing and standardization on the system after completing one-time detection. All sensor response data were evaluated based on the G/GO ratio (G represents the conductivity of the 14 sensors monitoring the sample gas and GO represents clean air). The environmental chamber is controlled as follows: room temperature (25 ℃); the cleaning flow of the sensor is 6L/min; automatic zero setting time is 10 s; both internal and inlet flow rates were 600 mL/min. The absorbed gas was measured every second during 60 seconds of the sample detection time. Each sample was analyzed in triplicate.
The comparison of the fragrance profile of the electronic nose was made between example 6 and comparative example 1, as shown in FIG. 3 (A: non-polar column DB-5; B: medium-polar column DB-1701).
The results are as follows: the two milk fans have certain difference on the whole aroma profile, and compared with the milk fan prepared by traditional acid water, the milk fan sample prepared by fermenting the acid water by using a leavening agent has obviously increased peak areas at a plurality of peak positions, which shows that the contents of various aroma substances and the whole aroma intensity thereof are improved. The result shows that the milk fan leavening agent can effectively enhance the whole fragrance of the milk fan.
The above-described embodiments are only preferred embodiments of the present invention, and are not intended to limit the present invention in any way and substantially, it should be noted that those skilled in the art may make several modifications and additions without departing from the scope of the present invention, which should also be construed as a protection scope of the present invention.
SEQUENCE LISTING
<110> Shanghai applied technology university
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Claims (10)
1. A milk fan fermentation strain with flavoring function is characterized by being at least one of lactobacillus plantarum ML9 and rhodotorula mucilaginosa 2-3;
the lactobacillus plantarum ML9 has a latin name of Lactplantibacillus plantarum ML9 and a preservation number of CCTCC M2021745, is preserved in China center for type culture Collection, and is preserved in Wuchang district, Wuhan City, Hubei province in eight Lou of Wuchang, and the preservation date is 2021 year, 6 months and 23 days.
The Latin name of the Rhodotorula mucilaginosa 2-3 is Rhodotorula mucor 2-3, the preservation number is CCTCC M2021747, the preservation unit is China center for type culture Collection, the preservation address is eight Lopa Alojia mountain in Wuchang district, Wuhan City, Hubei province, and the preservation date is 2021 year, 6 months and 23 days.
2. Use of a strain of fermented milk fan with flavouring function according to claim 1 for the preparation of a starter for milk fan.
3. A fermenting agent for milk fan with fragrance increasing function, which is characterized in that the fermenting agent is acid water fermented by the fermenting strain for milk fan with fragrance increasing function as claimed in claim 1.
4. The milk fan leavening agent with a flavoring function according to claim 3, wherein the milk fan leavening agent comprises acid water A fermented by lactobacillus plantarum ML9 and acid water B fermented by rhodotorula mucilaginosa 2-3.
5. The flavor-enhancing milk fan leavening agent according to claim 4, wherein the volume ratio of the acid water A to the acid water B is 1: 1.
6. The milk fan leavening agent with the flavor enhancement function according to any one of claims 3 to 5, wherein the preparation method of the milk fan leavening agent comprises the following steps:
step 1: preheating raw milk, adding traditional acid water, continuously heating until the milk is solidified;
step 2: filtering out the coagulated block by using a filter screen, and obtaining new making acid water by remaining whey;
and step 3: preparing acid water, inoculating lactobacillus plantarum ML9, and fermenting at room temperature to obtain acid water A;
and 4, step 4: preparing fresh acid water, inoculating Rhodotorula mucilaginosa 2-3, and fermenting at room temperature to obtain acid water B;
and 5: mixing the acid water A and the acid water B to obtain the dairy fan starter.
7. The milk fan leavening agent with flavor enhancement function according to claim 6, wherein the temperature of preheating and continuous heating in step 1 of the preparation method are both 65 ℃; the mass ratio of the raw milk to the traditional acid water is 1: 1.
8. The flavor-enhancing milk fan starter culture according to claim 6, wherein the inoculation concentration of Lactobacillus plantarum ML9 in step 3 of the preparation method is 2.0-2.8 x 104CFU/mL, and the fermentation time is 5-6 days.
9. The flavor-enhancing milk fan starter culture according to claim 6, wherein the inoculation concentration of Rhodotorula mucilaginosa 2-3 in step 4 of the preparation method is 5.2-6.0 x 105CFU/mL, and the fermentation time is 5-6 days。
10. Use of the dairy fan starter culture with a flavoring function of any one of claims 3 to 5 in the preparation of dairy fans.
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| WO2012159922A1 (en) * | 2011-05-26 | 2012-11-29 | Dsm Ip Assets B.V. | Process for manufacturing of a fermented dairy product |
| CN107974420A (en) * | 2017-12-17 | 2018-05-01 | 石家庄君乐宝乳业有限公司 | Lactobacillus bulgaricus JMCC0018, its isolation and purification method and the application of high yield acetaldehyde |
| CN109971871A (en) * | 2019-03-27 | 2019-07-05 | 江南大学 | A kind of method and its application screened and/or identify lactobacillus |
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| WO2012159922A1 (en) * | 2011-05-26 | 2012-11-29 | Dsm Ip Assets B.V. | Process for manufacturing of a fermented dairy product |
| CN107974420A (en) * | 2017-12-17 | 2018-05-01 | 石家庄君乐宝乳业有限公司 | Lactobacillus bulgaricus JMCC0018, its isolation and purification method and the application of high yield acetaldehyde |
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