Lactobacillus bulgaricus LYJ-9 and application thereof in preparation of cheese-flavored yoghourt

Abstract

The invention belongs to the field of microbial fermentation, and particularly relates to lactobacillus bulgaricus LYJ-9 and application thereof in preparation of cheese-flavored yoghourt. The preservation number of the lactobacillus bulgaricus LYJ-9 is CCTCC (China Center for Type Culture Collection) NO: M 20242587, the lactobacillus bulgaricus LYJ-9 is classified and named as Lactobacillus bulgaricus LYJ-9, the preservation date is November 18, 2024, and the preservation unit is China Center for Type Culture Collection. The lactobacillus bulgaricus LYJ-9 provided by the invention can endow the fermented milk with an obvious cheese flavor after fermentation, and the fermented milk obtained by compounding the lactobacillus bulgaricus LYJ-9 with streptococcus thermophilus not only has the cheese flavor, but also has a good level on the conventional quality of yoghourt. When the lactobacillus bulgaricus LYJ-9 provided by the invention is used for yoghourt fermentation, no auxiliary materials need to be added externally to enhance the flavor, the problem of yoghourt flavor homogenization is solved from the source of a fermented milk strain, and the requirements of consumers on health are met.

Description

Technical Field

[0001] The present invention belongs to the field of microbial fermentation, and specifically relates to Lactobacillus bulgaricus LYJ-9 and its application in the preparation of cheese-flavored yogurt. Background Art

[0002] Yogurt is generally produced by the mixed fermentation of Lactobacillus bulgaricus subsp. bulgaricus and Streptococcus thermophilus. With the continuous expansion of the market scale of the yogurt industry, various flavored yogurt products have emerged in an endless stream. However, the problem of homogenization of yogurt flavors has become increasingly prominent. Except for the sensory changes brought about by the additional fruit flavors or other auxiliary materials, consumers feel that the flavors of most yogurts are gradually converging. There are a wide variety of fermented milks, and flavor plays an important role in determining consumers' acceptance and preference for fermented milks. Compared with plain fermented milk, products that generally choose to add auxiliary materials to enhance the flavor of fermented milk during production are more popular. However, due to the public's yearning for a healthy and green lifestyle, the externally added flavors can no longer meet consumers' demands for the natural flavor attributes of yogurt products.

[0003] At present, cheese aroma, as a unique smell, is becoming increasingly popular in China. The consumption of the cheese industry continues to grow, and the market scale shows a stable growth. Innovative categories using cheese as a food ingredient are also emerging on the market. Therefore, it becomes meaningful to develop fermented milk with cheese-like characteristic flavors without adding external flavoring auxiliary materials. Summary of the Invention

[0004] The present invention has discovered a Lactobacillus bulgaricus with an obvious cheese flavor in fermented milk, which can solve the problem of homogenization of yogurt flavors from the source of fermented milk strains. The specific technical solutions are as follows:

[0005] In the first aspect, the present invention provides Lactobacillus bulgaricus LYJ-9, the preservation number of Lactobacillus bulgaricus LYJ-9 is CCTCC NO: M 20242587, the taxonomic name is Lactobacillus bulgaricus LYJ-9, the preservation date is November 18, 2024, and the preservation unit is China Center for Type Culture Collection.

[0006] The present invention has discovered a strain of Lactobacillus bulgaricus LYJ-9, whose fermented milk has an obvious cheese flavor and a high sensory evaluation score. Through quantitative analysis, it is found that the contents of two key flavor substances, butyric acid and hexanoic acid, in its fermented milk are relatively high, which are the main sources of the cheese flavor.

[0007] In the second aspect, the present invention provides a bacterial agent for yogurt fermentation, and the bacterial agent includes the above-mentioned Lactobacillus bulgaricus LYJ-9.

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

[0009] Furthermore, the Streptococcus thermophilus is Streptococcus thermophilus S4-1-1.

[0010] Furthermore, the ratio of the Streptococcus thermophilus to Lactobacillus bulgaricus LYJ-9 is 1000 to 1:1.

[0011] After fermenting the mixture of Lactobacillus bulgaricus LYJ-9 and Streptococcus thermophilus, the obtained fermented milk not only has a significantly improved cheese flavor, but also has a level similar to that of common yogurt in terms of tissue morphology, color, taste, etc. Therefore, the compound bacterial agent of Lactobacillus bulgaricus LYJ-9 and Streptococcus thermophilus has good market prospects.

[0012] In the present invention, the reference source of the cheese flavor is Cheddar cheese. The yogurt prepared with the compound bacterial agent of Lactobacillus bulgaricus LYJ-9 and Streptococcus thermophilus can have an aroma intensity similar to that of Cheddar cheese at most, which can solve the problem of flavor homogenization of yogurt from the source of fermented milk strains, meet consumers' preference for yogurt flavor and the pursuit of "zero-additive" yogurt.

[0013] When using Lactobacillus bulgaricus LYJ-9 for fermentation, the required fermentation time is short, and after the pH reaches a certain level, the change range of the acidity of the fermented milk with time is very small, reducing the occurrence of post-acidification of the fermented milk. In the prior art, in order to control the post-acidification of the fermented milk, the proportion of Streptococcus thermophilus to Lactobacillus bulgaricus will be increased. Therefore, by using Lactobacillus bulgaricus LYJ-9 provided in the present application, the proportion of Lactobacillus bulgaricus in the compound strains can be increased, better playing the role of Lactobacillus bulgaricus LYJ-9 provided in the present application in providing cheese flavor, and the available proportion of Lactobacillus bulgaricus in the compound strains is larger, effectively controlling the intensity of the cheese flavor of yogurt.

[0014] In the third aspect, the present invention provides the application of the above-mentioned Lactobacillus bulgaricus LYJ-9 or the above-mentioned bacterial agent for yogurt fermentation in the preparation of cheese-flavored yogurt.

[0015] In the fourth aspect, the present invention provides a method for preparing cheese-flavored yogurt, comprising the following steps:

[0016] S1: Activate the Lactobacillus bulgaricus LYJ-9 as described in claim 1 or the bacterial agent as described in claim 3, add it to sterilized pure milk for fermentation, and stop fermentation when reaching the fermentation end point;

[0017] S2: Stop fermentation and ripen the fermentation system to obtain cheese-flavored yogurt.

[0018] Further, in S1, the proportion of Lactobacillus bulgaricus LYJ-9 added is 2%.

[0019] Further, in S1, white granulated sugar is added to the pure milk, and the mass-volume ratio of the white granulated sugar to the pure milk is 0-10%.

[0020] Further, in S1, during the fermentation process, the fermentation temperature is 40-44 °C.

[0021] Further, in S1, the pure milk is placed in a water bath at 60 °C for 30 min, then sterilized at a high temperature in a water bath at 95 °C for 5 min, quickly ice-bathed to about 42 °C, and then fermented by adding fermenting bacteria.

[0022] Further, in S2, the way of after-ripening is: storing the sample after fermentation in a refrigerator at 4 °C for after-ripening for 24 h.

[0023] In the fifth aspect, the present invention provides a cheese-flavored yogurt prepared by the above preparation method.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] The Lactobacillus bulgaricus LYJ-9 provided by the present invention can endow the fermented milk with an obvious cheese flavor after fermentation, and the fermented milk obtained by compounding with Streptococcus thermophilus not only has a cheese flavor, but also has a good level in the conventional quality of yogurt. Using the Lactobacillus bulgaricus LYJ-9 provided by the present application for yogurt fermentation does not require exogenous addition of auxiliary materials to enhance the flavor, solves the problem of yogurt flavor homogenization at the source of fermented milk strains, and meets the health needs of consumers. Description of the Drawings

[0026] Figure 1 It is a morphological diagram of LYJ-9 bacteria.

[0027] Figure 2 It is a pH change curve diagram of LYJ-9 fermented milk.

[0028] Figure 3 A sensory evaluation radar chart of compound fermented milk and commercial starter. Specific Embodiments

[0029] In order to enable those skilled in the art to better understand the solution of the present invention, the technical solution of the present invention will be clearly and completely described below in conjunction with specific embodiments. It should be noted that the following detailed description is exemplary and is only a part of the embodiments of the present invention, rather than all of the embodiments.

[0030] Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the scope of protection of the present invention.

[0031] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which this application belongs. The experimental materials used in the embodiments of the present invention are all conventional experimental materials in the art and can be obtained through commercial channels. The experimental methods without detailed conditions are carried out according to conventional experimental methods or according to the operation manuals recommended by the suppliers.

[0032] Isolation and identification of the strain in Example 1

[0033] 1. Isolation of the strain

[0034] Method for isolating Lactobacillus bulgaricus: Take 1 mL of the collected fermented milk and add it to physiological saline for gradient dilution. Take 100 μL of the dilution and spread it evenly on MRS solid medium, and culture it at 37 °C for 48 h. Select colonies with irregular shapes for Gram staining, and select colonies with rod-shaped morphology and inoculate them into MRS liquid medium, and culture them at 37 °C for 24 h. Perform plate streaking on MRS solid medium and repeat twice. Ensure that the Gram staining is a pure bacterium. Take the colonies into MRS liquid medium and culture them at 37 °C for 24 h. When the bacterial liquid becomes turbid and there is flocculent precipitate at the bottom of the test tube, perform strain identification.

[0035] 2. Strain identification

[0036] After Gram staining, the morphology of Lactobacillus bulgaricus LYJ-9 was observed under an optical microscope with an oil immersion objective of 100 times. The microscopic examination results of the strain are as Figure 1 shown. The strain was subjected to 16S rRNA sequencing (the results are shown in SEQ ID NO.1), and the sequencing results were compared for homology on NCBI using BLAST: The results showed that the 16S rRNA sequence of the strain was 99.9% similar to that of Lactobacillus bulgaricus, proving that the strain belongs to the genus Lactobacillus bulgaricus, but there are differences in some sequences, and it was named Lactobacillus bulgaricus LYJ-9.

[0037] SEQ ID NO.1:

[0038]

[0039] Preparation and Analysis of Fermented Milk in Example 2

[0040] Using pure milk as raw milk, add 6% (wt / v) granulated sugar, water bath at 60 °C for 30 min, then sterilize at high temperature in a water bath at 95 °C for 5 min, and quickly cool in an ice bath to about 42 °C. In a laminar flow hood, add the activated L. bulgaricus strain to the sterilized raw milk at a ratio of 2% and stir evenly, then place it in an incubator at 42 °C for fermentation. Stop fermentation when the pH of the fermented milk drops to 4.5, and cool the sample. Store the fermented sample in a refrigerator at 4 °C for after-ripening. After 24 h of after-ripening, it is ready for sensory analysis and instrumental analysis.

[0041] The activation method of the L. bulgaricus strain is as follows: inoculate the preserved L. bulgaricus strain into MRS broth at an inoculation amount of 2% (wt / v) under aseptic conditions, incubate at 37 °C for 24 h, and store at a constant temperature for later use.

[0042] (1) Evaluation of Cheese Aroma

[0043] Evaluate the cheese aroma intensity of the fermented milk of the single L. bulgaricus strain. The definitions of aroma attribute descriptors and reference objects are shown in Table 1, and the evaluation scoring basis is in accordance with Table 2.

[0044] Table 1 Definitions of Aroma Attribute Descriptors and Reference Objects

[0045]

[0046] Table 2 Scoring Basis for the Intensity of Aroma Attributes

[0047]

[0048] The results of the cheese aroma evaluation are shown in Table 1. It can be seen that compared with other strains, LYJ-9 shows a stronger cheese flavor.

[0049] Table 3 Statistical Results of the Cheese Aroma Intensity Evaluation of Fermented Milk of 12 L. bulgaricus Strains and Statistical Results of the Fermentation End Time of Fermented Milk

[0050]

[0051] (2) Analysis of Semi-Quantitative Results of Flavor Compounds by HS-SPME-GC-MS

[0052] Weigh 8.0 g of the fermented milk sample and put it into a headspace vial. Add 1 μL of 0.769 mg / mL 2-octanol solution as the internal standard. Place the headspace vial containing the sample and internal standard mixture into the solid-phase microextraction device, heat it at 55 °C for 20 min to reach equilibrium and simultaneously stir it magnetically at a rotation speed of 550 r / min. Insert the aged CAR / PDMS extraction fiber into the headspace above the headspace vial, and inject the sample after 40 min of headspace extraction. After the extraction is completed, insert the extraction fiber into the GC injection port for 6 min of desorption, and the desorption temperature is 250 °C. The chromatographic column is an Agilent DB-Wax capillary column (30 m × 0.25 mm, 0.25 μm). The carrier gas uses 99.99% high-purity helium gas, and its flow rate is 1 mL / min. Secondly, according to C 7 ~C 40 saturated n-alkanes (Sigma-Aldrich, Shanghai, China), calculate the retention index of volatile compounds on the HP-INNOWax chromatographic column and compare it with the literature data. At the same time, in order to construct a standard curve, HS-SPME-GC-MS was used to analyze and detect the flavorless mechanism samples of fermented milk containing standard samples of aroma-active compounds with different concentrations under the same conditions, and a standard curve was obtained. Substitute the loudness value in the sample to be measured into the standard curve to find the absolute content of the key differential compounds.

[0053] Adopt the internal standard method for semi-quantitative analysis, and use formula (1) to calculate the content of the compound in the fermented milk system:

[0054]

[0055] In the formula, X 1 is the content of the compound to be determined in the fermented milk system; X 0 is the content of the internal standard in the 8.0 g fermented milk system; S 1 is the integrated peak area of the compound; S 0 is the integrated peak area of the internal standard.

[0056] Use formula (2) to calculate the retention index RI of the compound:

[0057]

[0058] In the formula, n is the number of carbon atoms, t n is the retention time of the n-alkane with n carbon atoms; t n+1 is the retention time of the n+1 carbon atom n-alkane; t i is the retention time of compound i.

[0059] Use formula (3) to calculate the standard curve of the compound:

[0060]

[0061] In the formula, x is the concentration of the aroma compound to be measured, y is the peak area of the aroma compound to be measured, Ai is the peak area of the internal standard compound, Ci is the concentration of the internal standard compound, k is the slope of the standard curve, and b is the intercept of the standard curve.

[0062] The qualitative results of the aroma-active components in fermented milk are shown in Table 3.

[0063] Table 4 Qualitative results of aroma-active components in fermented milk

[0064]

[0065] Continued Table 4

[0066]

[0067] (3) Analysis of the quantitative results of flavor substances by HS-SPME-GC-MS

[0068] The content of key flavor substances was determined by HS-SPME-GC-MS. As can be seen from Table 4, butyric acid and hexanoic acid in LYJ-9 fermented milk contribute greatly to the aroma of fermented milk cheese.

[0069] Table 5 Qualitative analysis and OAV analysis results of organic acids in fermented milk

[0070]

[0071] Note: The quantitative results are expressed as mean ± standard deviation (SD); different letters in the same row indicate significant differences (p < 0.05).

[0072] (4) pH value change curve of LYJ-9

[0073] During the fermentation process, the pH of the yogurt was measured according to the operation of the national standard "GB 5009.239-2016 National Food Safety Standard Determination of Food Acidity". Among them, the pH value change curve of LYJ-9 was measured, and the results are as Figure 2 shown. It can be seen that LYJ-9 produces acid faster in the early stage of fermentation. When the pH reaches about 4.2, the acidity value changes relatively smoothly, and the acid production rate decreases.

[0074] Example 3 Co-fermentation of Lactobacillus bulgaricus LYJ-9 and Streptococcus thermophilus

[0075] (1) Fermentation indexes of different co-fermentation ratio groups

[0076] Lactobacillus bulgaricus LYJ-9 and Streptococcus thermophilus S4-1-1 (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) were compounded at ratios of 10:1 (based on the addition amount of Lactobacillus bulgaricus of 1×10 8 CFU / mL), 1:1, 1:10, 1:100, and 1:1000 (based on the addition amount of Streptococcus thermophilus of 1×10 8 CFU / mL) respectively to prepare fermented milk. The fermentation method was the same as that in Example 2. After obtaining the fermented milk, sensory evaluation was carried out. The results are shown in Tables 6 and 7. It can be seen that the cheese aroma intensity of the F2 compound fermented milk was higher with higher butyric acid and hexanoic acid contents, and the fermentation time was the shortest. Through comprehensive comparison, the final optimal compound ratio was determined as Lactobacillus bulgaricus:Streptococcus thermophilus = 1:1.

[0077] Table 6 Fermentation indexes of different compound ratio groups

[0078]

[0079] (2) Sensory evaluation comparison between the fermented milk with the optimal compound ratio and commercial starter

[0080] Sensory evaluation was carried out on F2 and commercial starter D1. The sensory evaluation criteria for fermented milk are shown in Table 7. It Figure 3 can be seen that there were no significant differences between F2 and D1 in terms of color, odor, taste, and tissue form, but the degree of preference of the evaluators for F2 was higher than that for D1.

[0081] Table 7 Sensory evaluation criteria for fermented milk

[0082]

Claims

1. Lactobacillus bulgaricus LYJ-9, characterized in that The preservation number of the bulgaricus Lactobacillus LYJ-9 is CCTCCNO: M 20242587, the classification name is Lactobacillus bulgaricus LYJ-9, the preservation date is November 18, 2024, and the preservation unit is the China Center for Type Culture Collection.

2. A bacterial agent for yogurt fermentation, characterized in that: The bacterial agent includes the Lactobacillus bulgaricus LYJ-9 described in claim 1.

3. The bacterial agent according to claim 2, characterized in that The bacterial agent also includes thermophilic streptococcus.

4. The bacterial agent according to claim 3, characterized in that The thermophilic streptococcus is thermophilic streptococcus S4-1-1.

5. Use of the Lactobacillus bulgaricus LYJ-9 according to claim 1, or the bacterial agent according to any one of claims 2 or 3 in preparing cheese-flavored yogurt.

6. A method for preparing cheese-flavored yogurt, characterized in that: The following steps are involved: S1: activating the Lactobacillus bulgaricus LYJ-9 according to claim 1 or the bacterial agent according to claim 3, adding the activated Lactobacillus bulgaricus LYJ-9 to sterilized pure milk for fermentation, and stopping the fermentation when the fermentation ends; S2: Stop the fermentation and post-ripen the fermentation system to obtain cheese-flavored yogurt.

7. The method for preparing cheese-flavored yogurt according to claim 6, characterized in that: In S1, the ratio of the Lactobacillus bulgaricus LYJ-9 to the pure milk is 2%.

8. The method for preparing cheese-flavored yogurt according to claim 6, characterized in that: In S1, white sugar is added to the pure milk, and the mass volume ratio of the white sugar to the pure milk is 0-10%.

9. The method for preparing cheese-flavored yogurt according to claim 6, characterized in that: In S1, during the fermentation process, the fermentation temperature is 40-44°C.

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

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