Lactococcus lactis YN18-6.2 and application of lactococcus lactis YN18-6.2 in preparation of nut flavor yoghourt

Abstract

The invention belongs to the field of microbial fermentation, and particularly relates to lactococcus lactis YN18-6.2 and application of the lactococcus lactis YN18-6.2 in preparation of nut-flavored yoghourt. The lactococcus lactis YN18-6.2 is classified and named as Lactococcus lactis YN18-6.2, the collection number of the lactococcus lactis YN18-6.2 is CCTCC M 20242586, the collection date of the lactococcus lactis YN18-6.2 is November 18, 2024, and the collection unit of the lactococcus lactis YN18-6.2 is China Center for Type Culture Collection. Lactococcus lactis YN18-6.2, streptococcus thermophilus and lactobacillus bulgaricus are subjected to compound fermentation, yoghourt with nut flavor can be obtained, and further analysis shows that a main substance influencing the flavor of a fermentation product of the strain is 3-methylbutyraldehyde; the compound bacterial agent of the lactococcus lactis YN18-6.2 disclosed by the invention has relatively good fermentation performance and relatively great market application potential.

Description

Technical Field

[0001] The invention belongs to the field of microbial fermentation, and particularly relates to Lactococcus lactis YN18-6.2 and application thereof in preparing nut-flavored yogurt. Background Art

[0002] Yogurt is a dairy product with high nutritional value and unique flavor. It has the benefits of maintaining the balance of gastrointestinal microflora, enhancing immunity, preventing osteoporosis, and lowering blood lipids and cholesterol, and is deeply favored by consumers. Its popularity is not only based on its prebiotic function, but also on its unique sensory properties. Among the sensory properties such as texture, color and flavor, flavor is an important factor affecting the quality of yogurt and plays an important role in consumer acceptance and preference.

[0003] Lactococcus lactis, Streptococcus thermophilus and Lactobacillus bulgaricus are the three most commonly used starter bacteria in the production of fermented dairy products, with a market value of billions of dollars. Lactococcus lactis plays a huge role in improving the texture and flavor of fermented dairy products by virtue of its ability to hydrolyze proteins and produce volatile flavor substances. It is widely used in the production of dairy products such as cheese, yogurt and sour cream, and has extremely high industrial and economic value. Lactococcus lactis has a certain enzyme that Lactobacillus delbrueckii subspecies bulgaricus and Streptococcus thermophilus do not have, and can produce specific flavor substances, thereby improving the flavor of yogurt.

[0004] With the growth of the yogurt market, consumers' demand for diversified yogurt flavors has become increasingly prominent, and the problem of yogurt flavor homogeneity has become increasingly serious. The root cause of yogurt homogeneity lies in the fermentation strain. Summary of the invention

[0005] In view of the problem of flavor homogeneity in current yogurt, the present invention provides Lactococcus lactis YN18-6.2 and its application in preparing nut-flavored yogurt. The specific technical scheme is as follows:

[0006] In a first aspect, the present invention provides Lactococcus lactis YN18-6.2, the classification name of Lactococcus lactis YN18-6.2 is Lactococcus lactis YN18-6.2, the preservation number is CCTCC M 20242586, the preservation date is November 18, 2024, and the preservation unit is China Center for Type Culture Collection.

[0007] In a second aspect, the present invention provides a bacterial agent for fermenting yogurt, wherein the bacterial agent comprises the Lactococcus lactis YN18-6.2 as described in claim 1.

[0008] Furthermore, the bacterial agent also includes Lactobacillus bulgaricus and Streptococcus thermophilus.

[0009] Furthermore, the Lactobacillus bulgaricus is Lactobacillus bulgaricus L9-1, and the Streptococcus thermophilus is Streptococcus thermophilus S6-13.

[0010] Furthermore, the ratio of Lactococcus lactis, Streptococcus thermophilus and Lactobacillus bulgaricus is 1-100:10:1.

[0011] Furthermore, the ratio of Lactococcus lactis, Streptococcus thermophilus and Lactobacillus bulgaricus is 100:10:1.

[0012] In a third aspect, the present invention provides the use of the above-mentioned Lactococcus lactis YN18-6.2, or the above-mentioned bacterial agent in the preparation of nut-flavored yogurt.

[0013] In a fourth aspect, the present invention provides a method for preparing nut-flavored yogurt, comprising the following steps:

[0014] The above-mentioned bacterial agent is activated, added into sterilized pure milk for fermentation, and the fermentation is stopped when the fermentation end point is reached, and the milk is post-ripened to obtain nut-flavored yogurt.

[0015] Furthermore, the volume ratio of the bacterial agent to pure milk is 2 to 3.5:100.

[0016] Furthermore, the volume ratio of the bacterial agent to pure milk is 2.5:100.

[0017] Furthermore, white sugar is added to the pure milk, and the mass volume ratio of the white sugar to the pure milk is 0 to 10%.

[0018] Furthermore, the mass volume ratio of the white sugar to the pure milk is 6%.

[0019] Furthermore, in S1, during the fermentation process, the fermentation temperature is 27-42°C.

[0020] Furthermore, the fermentation temperature was 32°C.

[0021] In a fifth aspect, the present invention provides cheese-flavored yogurt prepared by the above-mentioned preparation method.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] This patent has discovered a nut-flavored Lactococcus lactis YN18-6.2. By compounding Lactococcus lactis YN18-6.2 with thermophilic Streptococcus and Lactobacillus bulgaricus for fermentation, a nut-flavored yogurt can be obtained, and further analysis shows that the main substance affecting the flavor of the fermentation product of this strain is 3-methylbutyraldehyde; the compound bacterial agent of Lactococcus lactis YN18-6.2 in this application has good fermentation performance and has great market application potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a radar chart of the aroma typing evaluation results of 28 strains of Lactococcus lactis complex fermented milk. The left one is the radar chart of the nut aroma evaluation results, and the right one is the radar chart of the malt aroma evaluation results.

[0025] Figure 2 Schematic diagram of multivariate statistical analysis of nutty aroma, malt flavor and control fermented milk components.

[0026] Figure 3 Schematic diagram of multivariate statistical analysis of nutty aroma, malt flavor and control fermented milk components.

[0027] Figure 4 This is the lactic acidification curve of the combined fermentation of Lactococcus lactis.

[0028] Figure 5 Schematic diagram of the effect of different sugar addition amounts on flavor acceptance.

[0029] Figure 6 Schematic diagram of the effect of different temperatures on flavor acceptance.

[0030] Figure 7 Schematic diagram of the effect of different strain ratios on flavor acceptance.

[0031] Figure 8 Schematic diagram of the effect of different inoculation volumes on flavor acceptance.

[0032] Fig. 9 Schematic diagram of the effect of different raw materials on flavor acceptance.

[0033] Fig.10 This is a schematic diagram of the viscosity and water separation rate analysis of Lactococcus lactis complex fermented milk; the left figure is a schematic diagram of viscosity analysis, and the right figure is a schematic diagram of water separation rate analysis.

[0034] Fig.11 This is a schematic diagram of the sensory analysis of Lactococcus lactis complex fermented milk; the left figure is a schematic diagram of the sensory score, and the right figure is a schematic diagram of the sensory acceptance score. DETAILED DESCRIPTION

[0035] In order to make those skilled in the art better understand the scheme of the present invention, the technical scheme of the present invention is clearly and completely described below in conjunction with specific embodiments. It should be noted that the following detailed descriptions are all exemplary and are only embodiments of a part of the present invention, rather than all embodiments.

[0036] Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative work should fall within the scope of protection of the present invention.

[0037] Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those of ordinary skill in the art to which this application belongs. The experimental materials used in the examples of the present invention are all conventional experimental materials in the art and can be purchased through commercial channels. Experimental methods without specifying detailed conditions were performed according to conventional experimental methods or according to the operating instructions recommended by the supplier.

[0038] Example 1 Isolation and Identification of Lactococcus lactis

[0039] The lactococcus lactis used in this case was isolated from fermented cow milk and sour camel milk. After Gram staining of the isolated strain, it was observed under an oil microscope and found that the lactococcus lactis was purple, which was a Gram-positive strain; under the oil microscope, the bacterial cells were round or oval, and formed chains along the long axis; most of them were in pairs or short chains, and some formed long chains, which was consistent with the description of lactococcus lactis in the Bergey's Manual of Bacterial Identification. It was preliminarily determined that the isolated strain was lactococcus lactis. Subsequently, 16S identification and classification were performed, and the sequencing results were compared to find that the strain used in the experiment was Lactococcus lactis.

[0040] Table 1 Strain sources and identification information

[0041]

[0042] Example 2 Preparation and Analysis of Fermented Milk

[0043] (1) The preparation of fermented milk comprises the following specific steps: pure milk is used as raw material milk, 6% (wt / v) white sugar is added, the mixture is heated in a water bath at 60°C for 30 minutes, and then sterilized in a water bath at 95°C for 5 minutes, and then quickly cooled to about 42°C in an ice bath. In the clean bench, activated Lactococcus lactis, Lactobacillus bulgaricus L9-1 (Ao L, Qiqi L, Yushan B, et al. Aroma classification and characterization of Lactobacillus delbrueckii subsp. bulgaricus fermented milk [J]. Food Chemistry: X, 2022, 15100385-100385.), and Streptococcus thermophilus S6-13 (Ao L, Hongwei Z, Tongjie L, et al. Aroma classification and flavor characterization of Streptococcus thermophilus fermented milk by HS-GC-IMS and HS-SPME-GC-TOF / MS [J]. Food Bioscience, 2022, 49) (strain ratio: 100: 10: 1) were added to the sterilized raw milk at a ratio of 2%, stirred evenly, and placed in a 42°C incubator for fermentation. When the pH of the fermented milk dropped to 4.5, the fermentation was stopped and the sample was cooled. The samples after fermentation were stored in a refrigerator at 4°C for post-ripening. After 24 hours of post-ripening, they were prepared for sensory analysis and instrumental analysis.

[0044] (2) Aroma classification evaluation

[0045] The aroma typing of fermented milks made from 28 different strains of Lactococcus lactis was evaluated. The definitions and references of the four aroma attribute descriptors are shown in Table 2. The evaluation scores are based on Table 3. The aroma typing results are shown in Table 4 and Table 5. Figure 1 , YN18-6.2 exhibited a stronger nutty flavor compared to the other strains.

[0046] Table 23 definitions and references of aroma attribute descriptors

[0047]

[0048] Table 3 Basis for scoring the intensity of aroma attributes

[0049]

[0050]

[0051] Table 4 Evaluation results of aroma typing of 28 strains of Lactococcus lactis mixed fermented milk

[0052]

[0053] Analogous to the aroma classification method of liquor, it is proposed that the aroma attribute with the highest sensory evaluation score, which is the prominent flavor, is defined as the aroma type. According to the sensory analysis results of fermented milk, 28 strains of Lactococcus lactis fermented milk were divided into 3 aroma types. The results are shown in Table 5, namely, nut aroma type, malt aroma type, and mixed aroma type. When the scores of nut flavor and malt flavor are the same, it is defined as mixed aroma type. Among them, there are 14 fermented milk samples with nut aroma, 9 fermented milk samples with malt aroma, and 5 fermented milk samples with mixed aroma.

[0054] Table 5 Typing results of 28 strains of Lactococcus lactis

[0055]

[0056] (3) HS-SPME-GC-MS analysis

[0057] 8.0g fermented milk sample was weighed and placed in a headspace bottle, and 5μL 0.8093mg / mL 2-methyl-3-heptanone solution was added as an internal standard. The headspace bottle containing the sample and internal standard mixture was placed in a solid phase microextraction device, heated and balanced at 55℃ for 20min and magnetically stirred at a speed of 600r / min. The aged CAR / PDMS extraction head was inserted into the headspace bottle, and the sample was injected after 40min of headspace extraction. After the extraction was completed, the extraction fiber was inserted into the GC injection port for 6min of analysis at a temperature of 250℃. The chromatographic column was an Agilent DB-Wax capillary column (30m×0.25mm, 0.25μm). The front injection port adopted a splitless injection mode, and the solvent delay was set to 3.2min. The carrier gas used 99.99% high-purity helium with a flow rate of 1mL / min. The interface was 250℃, the connecting rod was 150℃, the ion source was 230℃, and the electron energy was 70eV. The mass spectrometer scanning range is: m / z 30~350.

[0058] The internal standard method was used in a semi-quantitative manner, and the content of the compound in the fermented milk system was calculated using formula (1):

[0059]

[0060] Wherein, X1 is the content of the desired compound in the fermented milk system; X0 is the content of the internal standard in 8.0 g of the fermented milk system; S1 is the integrated peak area of ​​the compound; S0 is the integrated peak area of ​​the internal standard.

[0061] The retention index RI of the compound was calculated using formula (2):

[0062]

[0063] Wherein, n is the number of carbon atoms, tn is the retention time of the normal alkane with the carbon number n; tn+1 is the retention time of the normal alkane with the carbon number n+1; and ti is the retention time of compound i.

[0064] The compound fermented milks of YN18-6.2 (nut flavor) and YN17-3.5 (malt flavor) obtained in step (1) and the fermented milk of the control group (Lactobacillus bulgaricus L9-1, Streptococcus thermophilus S6-13) were selected for analysis. The results are shown in Table 6. It can be seen that a total of 15 ketones, 4 aldehydes, 13 alcohols, 21 organic acids, 6 esters, 3 sulfur-containing compounds, and 6 other compounds were identified.

[0065] Table 6 Types of volatile compounds in fermented milk with different aromas

[0066]

[0067]

[0068] (4) HS-SPME-GC-MS determination of different compounds in fermented milk with nut aroma

[0069] OPLS-DA is a multivariate correction analysis method used to analyze the correlation between data sets after PCA dimensionality reduction. OPLS-DA analysis was performed on GC-MS data, and nut-flavor fermented milk was compared with malt-flavor and control groups for OPLS-DA analysis to screen differential volatile compounds. Two OPLS-DA models were generated by comparing the nut-flavor and other fermented milk samples in the three fermented milk samples. Figure 2 As shown in the figure, it can be found that the fermented milk sample YN18-6.2 is distributed on the right side of the axis, and the YN17-3.5 and control groups are distributed on the left side of the axis. The three parallel samples can be well separated from the fermented milk samples. Figure 3 As shown in the figure, after 200 permutation tests, R2 and Q2 are both greater than 0.5, indicating that the model does not have overfitting phenomenon and the model construction is effective and feasible. PCA combined with OPLS-DA illustrates the accuracy of the results.

[0070] By setting the screening conditions: VIP value>1.0, p<0.05 and FDR value<0.05, a total of 8 differential volatile flavor compounds were screened out in the first model YN16-6.2 and YN17-3.5 (Table 8). Similarly, in the second model YN16-6.2 and the control group, 5 differential compounds were screened out, as shown in Table 7. In the two models, a total of 3 differential compounds were found, namely 3-methylbutanal, isopentanol, and phenylethanol.

[0071] Table 7 VIP calculation for pairwise comparison between nut-flavored fermented milk and control group fermented milk

[0072]

[0073]

[0074] Table 8 VIP calculation for pairwise comparison between nut-flavored fermented milk and malt-flavored fermented milk

[0075]

[0076] (5) Quantitative analysis of aroma compounds using the external standard method using HS-SPME-GC-MS

[0077] The contents of key flavor substances were determined by HS-SPME-GC-MS external standard method. The results are shown in Table 9.

[0078] As can be seen from Table 9, 3-methylbutanal was not detected in the control group (Thermophilus Streptococcus and Lactobacillus bulgaricus) of malt-flavored milk and milk fermented without Lactococcus lactis. YN18-6.2 compound fermented milk contains 3-methylbutanal. Although the content is low, the threshold value of this aldehyde is low and it is easier to be perceived at low concentrations.

[0079] Table 9 Standard curve of volatile compounds and quantitative results of external standard method

[0080]

[0081] Note: 1. 2-Octanone was used as internal standard; 2. Ions were selected in SIM mode; 3. Quantitative ions; 4. Quantitative results were expressed as mean ± standard deviation (SD); Different letters in the same row indicated significant differences (p<0.05).

[0082] Table 10 Calculation results of OAV of volatile compounds

[0083]

[0084]

[0085] Table 11 Sensory evaluation results of aroma addition experiment

[0086]

[0087] Taking the control group as a reference, the addition experiment was carried out according to the quantitative results of the external standard method; (difference test and similarity sensory score) After verification by the aroma addition experiment, 3-methylbutanal was found to be the most critical aroma compound affecting the aroma of nuts.

[0088] Example 3 Acidification of Lactococcus lactis compound fermented milk

[0089] Lactococcus lactis YN18-6.2 was used for fermentation alone, Streptococcus thermophilus and Lactobacillus bulgaricus were used for fermentation together, and Streptococcus thermophilus, Lactobacillus bulgaricus, and Lactococcus lactis YN18-6.2 were used for fermentation together. The fermentation method was the same as that in Example 2, and the pH change during the fermentation process was detected. The results are as follows: Figure 4 shown.

[0090] Depend on Figure 4 It can be seen that the pH value decreases with the extension of time. Compared with the milk fermented by Lactococcus lactis YN18-6.2 alone, the fermented milk with three strains significantly accelerated the acid production rate and shortened the fermentation time. On the one hand, it may be due to the interaction between the strains to accelerate the acid production. On the other hand, it may be due to the faster acid production rate of the strain in the control group, which also has a certain effect on the acid production of the compound fermented milk.

[0091] Example 4 Fermentation process analysis of Lactococcus lactis YN18-6.2 compound fermented milk

[0092] (1) Analysis of sugar addition

[0093] The fermentation of the Lactococcus lactis YN18-6.2 compound fermented milk was carried out according to the fermentation method of Example 2 with 0%, 2%, 4%, 6% and 8% sugar addition. The sensory analysis results are as follows: Figure 5 As shown, it can be seen that the sensory acceptance is the highest under the condition of 6% sugar addition, and there is no significant difference in flavor with the addition of 2%, 4%, and 6%, and there is a significant difference with the condition of no sugar addition (p<0.05). As the amount of sugar added increases, the sensory acceptance decreases, which may be because the sweetness is too high, affecting the taste of fermented milk.

[0094] (2) Fermentation temperature analysis

[0095] The fermentation temperature was 27°C, 32°C, 37°C, and 42°C, and the fermentation method of Example 2 was used to ferment the Lactococcus lactis YN18-6.2 compound fermented milk. The sensory analysis results were as follows: Figure 6 As shown, it can be seen that the sensory acceptance at 32°C is the highest, which is close to the score at 42°C. Lactococcus lactis is a mesophilic lactic acid bacterium, and the fermentation time at 42°C is longer, so 32°C is the optimal fermentation temperature.

[0096] (3) Strain ratio analysis

[0097] The fermentation was carried out according to the ratio of Lactococcus lactis YN18-6.2, Streptococcus thermophilus and Lactobacillus bulgaricus of 1:10:1, 10:10:1, 10:10:1 and 1000:10:1. The sensory analysis results are as follows: Figure 7As shown, it can be seen that when the ratio of Lactococcus lactis: Streptococcus thermophilus: Lactobacillus bulgaricus is 100:10:1, the sensory acceptance is the highest, and it is significantly different from other ratios.

[0098] (4) Inoculation volume analysis

[0099] The fermentation of Lactococcus lactis YN18-6.2 compound fermented milk was carried out with an inoculation volume of 2%, 2.5%, 3%, 3.5%, and 4%. The sensory analysis results are as follows Figure 8 As shown, it can be seen that 3% has the highest sensory acceptance, followed by 2.5%, but there is no significant difference in the sensory acceptance scores between the two.

[0100] (5) Raw material analysis

[0101] The sensory analysis results of the fermented milk prepared by Lactococcus lactis YN18-6.2 were as follows: Fig. 9 As shown, it can be seen that the fermented milk prepared from whole milk has a higher score, which is significantly different from the scores of other raw materials.

[0102] (6) Fermentation based on the results of the above fermentation process analysis

[0103] The fermentation conditions were as follows: temperature 32℃, sugar addition 6%, inoculation 2.5%, strain ratio (Lactococcus lactis YN18-6.2: Streptococcus thermophilus: Lactobacillus bulgaricus) 100:10:1, and whole milk as raw material, as compound fermented milk; Lactococcus lactis YN18-6.2 was used alone as single fermented milk; Streptococcus thermophilus and Lactobacillus bulgaricus were used as fermentation bacteria as control fermented milk, in which the ratio of Streptococcus thermophilus and Lactobacillus bulgaricus fermentation group was 10:1. The quality analysis results of fermented milk are as follows: Fig.10 The sensory analysis results are shown in Fig.11 shown.

[0104] Depend on Fig.10 It can be seen that in terms of viscosity, the control group has the highest viscosity, followed by the compound fermented milk, and the single strain has the lowest viscosity, with significant differences. The water separation rate of the compound fermented milk is between the control fermented milk and the single fermented milk.

[0105] Depend on Fig.11 It can be seen that the texture sensory score of the compound fermented milk is the highest, while the texture of the control fermented milk is second, which means that the high viscosity of the control fermented milk will reduce the sensory personnel's liking for it. In the evaluation of flavor sensory acceptance, it was found that the score of the fermented milk alone was the lowest. This may be due to the high content of 3-methylbutanal, which caused the transformation of the nutty flavor into another flavor and reduced the sensory personnel's liking for it. Compared with the control fermented milk, the compound fermented milk has a higher sensory score.

Claims

1. Lactococcus lactis YN18-6.2, characterized in that The classification name of the Lactococcus lactis YN18-6.2 is Lactococcus lactis YN18-6.2, the preservation number is CCTCC M 20242586, the preservation date is November 18, 2024, and the preservation unit is the China Center for Type Culture Collection.

2. A bacterial agent for fermenting yogurt, characterized in that: The bacterial agent includes the Lactococcus lactis YN18-6.2 as described in claim 1.

3. The bacterial agent according to claim 2, characterized in that The bacterial agent also includes Lactobacillus bulgaricus and Streptococcus thermophilus.

4. The bacterial agent according to claim 3, characterized in that The Lactobacillus bulgaricus is Lactobacillus bulgaricus L9-1, and the Streptococcus thermophilus is Streptococcus thermophilus S6-13.

5. Use of the Lactococcus lactis YN18-6.2 according to claim 1, or the bacterial agent according to any one of claims 2 to 4 in the preparation of nut-flavored yogurt.

6. A method for preparing nut-flavored yogurt, characterized in that: The following steps are involved: The bacterial agent as claimed in claim 4 is activated, added into sterilized pure milk for fermentation, and the fermentation is stopped when the fermentation end point is reached, and the milk is post-ripened to obtain nut flavored yogurt.

7. The method for preparing nut-flavored yogurt according to claim 6, characterized in that: The volume ratio of the bacterial agent to pure milk is 2 to 3.5:

100.

8. The method for preparing nut-flavored yogurt according to claim 6, characterized in that: White sugar is added into the pure milk, and the mass volume ratio of the white sugar to the pure milk is 0-10%.

9. The method for preparing nut-flavored yogurt according to claim 6, characterized in that: In S1, during the fermentation process, the fermentation temperature is 27-42°C.

10. Cheese-flavored yogurt prepared by the preparation method according to any one of claims 6 to 9.

Images