Kluyveromyces marxianus and application of Kluyveromyces marxianus in preparation of rose fruity fermented milk

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to kluyveromyces marxianus and application thereof in preparation of rose fruity fermented milk. The Kluyveromyces marxianus strain is named as ALE-X20, is preserved in the China General Microbiological Culture Collection Center (CGMCC), is classified and named as Kluyveromyces marxianus, has the preservation number of CGMCC NO.32221, and is preserved on October 15, 2024. The Kluyveromyces marxianus ALE-X20 disclosed by the invention can efficiently convert an amino acid substrate into a key flavor substance, namely, 2-phenethyl alcohol, of rose fruit fragrance, so that the unique rose fragrance is given to the fermented milk under the condition that no essence or spice is added.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to Kluyveromyces marxianus and application thereof in preparing rose fruit-flavored fermented milk. Background Art

[0002] Kluyveromyces marxianus belongs to the genus Kluyveromyces. Kluyveromyces marxianus is listed as an edible species and can be added to fermented foods. Patent CN201510527940.0 discloses a new strain of Kluyveromyces marxianus that can be used to prepare red tea protein beverages and lactose hydrolysis. Studies have also shown that when milk-derived Kluyveromyces marxianus is used as a strain for milk beer fermentation, it can partially or completely replace brewer's yeast or brewer's yeast for milk beer production. Patent CN201810186371.1 discloses a freeze-dried milk beer starter of a composite of Kluyveromyces marxianus and brewer's yeast. Patent CN201710355851.1 discloses a preparation method and use of a freeze-dried bacterial agent of Kluyveromyces marxianus.

[0003] The above study shows that it is feasible to ferment beverages by combining Kluyveromyces marxianus with other strains, but the above document does not disclose the effect of Kluyveromyces marxianus on milk flavor substances and sensory properties. The prior art lacks acid-resistant Kluyveromyces marxianus that can add new flavors and sensory properties to fermented beverages.

[0004] The fermented milk products on the market currently have the core problems of single starter type and serious flavor homogeneity. In addition, the aroma of the floral and fruity flavored fermented milk on the market comes from edible flavors, which are mainly produced by chemical synthesis. Although the appropriate addition of flavors is beneficial and harmless, traditional consumer concepts still tend to prefer clean food labels. Most of the existing technologies are based on the selection and regulation of the ratio of starters. For example, the composite starter of Kluyveromyces marxianus and lactic acid bacteria has been used in the food fermented milk system. There is a lack of technology to optimize the fermentation substrate based on the improvement of the starter.

[0005] Currently, the Kluyveromyces marxianus on the market is mainly used for wine production, but is rarely used in fermented milk systems. The main reason is that the growth of Kluyveromyces marxianus will be inhibited in an acidic environment. The existing technology lacks acid-resistant domestication technology for Kluyveromyces marxianus, and the existing strains cannot maintain stable growth and aroma metabolism in the acid environment of the fermented milk system. Summary of the invention

[0006] In order to overcome the problems of the prior art, the present invention proposes a Kluyveromyces marxianus and its application in preparing rose fruit aroma fermented milk. The Kluyveromyces marxianus can efficiently convert amino acid substrates into 2-phenylethanol, a key flavor substance of rose fruit aroma, thereby giving the fermented milk a unique rose aroma without adding any essence or fragrance.

[0007] The object of the present invention is achieved in that:

[0008] A first aspect of the present invention provides a Kluyveromyces marxianus, wherein the Kluyveromyces marxianus is named ALE-X20, and the strain is deposited in the General Microbiological Center of China Microorganism Culture Collection Administration, and is classified and named Kluyveromyces marxianus, with a deposit number of CGMCC NO.32221 and a deposit date of October 15, 2024.

[0009] Furthermore, the Kluyveromyces marxianus has good growth and metabolism conditions under acidic conditions of pH 4.0-6.0.

[0010] The second aspect of the present invention provides a bacterial agent, comprising the Kluyveromyces marxianus described in the first aspect.

[0011] The third aspect of the present invention provides a composite probiotic agent, wherein the composite probiotic agent comprises Kluyveromyces marxianus and Lactococcus lactis as described in the first aspect;

[0012] The lactococcus lactis includes Lactococcus lactis subsp. cremoris and Lactococcus lactis subsp. lactis.

[0013] Furthermore, the two strains of bacteria are mixed in a ratio of 1:10 to 1:30 in terms of live bacteria content.

[0014] The fourth aspect of the present invention provides a product prepared from the Kluyveromyces marxianus described in the first aspect, or the bacterial agent described in the second aspect, or the composite probiotic bacterial agent described in the third aspect, wherein the product is any one of food, health care product, and medicine.

[0015] The fifth aspect of the present invention provides the use of the Kluyveromyces marxianus described in the first aspect, or the bacterial agent described in the second aspect, or the composite probiotic bacterial agent described in the third aspect in the preparation of a rose fruit-flavored fermented milk product.

[0016] Furthermore, the application is to increase the content of characteristic flavor substances including 2-phenylethanol, ethyl acetate and isopentanol during the fermentation process, thereby giving the fermented milk product a rose fragrance.

[0017] The sixth aspect of the present invention provides a method for preparing rose fruit-flavored fermented milk, comprising the following steps:

[0018] (1) Preparation of raw milk: Add water to the raw milk powder and stir thoroughly to dissolve and hydrate; the hydration conditions are hydration at 35°C-50°C for 0.5-1h, or hydration below 6°C for 8-12h;

[0019] (2) adding amino acids to the hydrated raw milk obtained in step (1), and sterilizing the raw milk at a temperature of 85° C. to 95° C. for 5 to 15 minutes;

[0020] Among them, the amount of amino acid added is 1 to 5 g / 1L raw milk;

[0021] (3) Adding the activated Kluyveromyces marxianus described in the first aspect or the composite probiotic agent described in the third aspect to the sterilized raw milk, with an inoculation amount of 1% to 10%; the inoculation amount is more preferably 1% to 5%;

[0022] (4) The prepared sample is fermented at 30±5°C for 12 to 36 hours, or at 40 to 42°C for 4 to 6 hours, then cooled to 30±5°C and continued to ferment for 12 to 24 hours; stirring is performed intermittently during the fermentation period, and then cooled and stored to obtain the rose fruit flavored fermented milk.

[0023] Furthermore, in step (2), the amino acid is phenylalanine.

[0024] Strain deposit information:

[0025] The Kluyveromyces marxianus was named ALE-X20. The strain was deposited in the General Microbiology Center of China Culture Collection Administration (CGMCC), address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing; the classification name is Kluyveromyces marxianus, the preservation number is CGMCC NO.32221, and the preservation date is October 15, 2024.

[0026] The advantages and beneficial effects of the present invention are:

[0027] 1. The present invention obtains a new strain of Kluyveromyces marxianus ALE-X20 through adaptive laboratory evolution. The strain has good growth and metabolism under acidic conditions of pH 4.0-6.0 and exhibits good acid resistance. The strain can efficiently convert amino acid substrates into 2-phenylethanol, a key flavor substance of rose fruit aroma, thereby giving fermented milk a unique rose aroma without adding any essence or fragrance.

[0028] 2. The present invention applies a composite probiotic agent consisting of Kluyveromyces marxianus ALE-X20 and Lactococcus lactis to the preparation of fermented milk. The two strains of bacteria form a complex promoting effect. While the content of 2-phenylethanol, a representative substance of rose fragrance, is increased, the content of isopentanol that imparts cream fragrance and ethyl acetate that imparts fruity fragrance are both increased, further giving the fermented milk product a rose fruit fragrance and improving the flavor of the fermented milk. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The present invention will be further described below in conjunction with the accompanying drawings and embodiments.

[0030] Figure 1 This is a simplified flow chart of the process optimization of rose fruit-flavored fermented milk according to Example 1 of the present invention;

[0031] Figures 2 to 4 This is a diagram of sensory QDA analysis results of the rose fruit flavor fermented milk process optimization in Example 1 of the present invention; wherein, Figure 2 The optimization of colony count and fermentation temperature is shown. Figure 3 The optimization of bacterial inoculum and amino acid addition is shown. Figure 4 The optimization of the ratio of double bacteria compound is shown;

[0032] Figure 5 is the design diagram of the flavoromics experiment described in the test example of the present invention;

[0033] Figure 6 This is a PCA analysis result diagram of four samples in the test example of the present invention;

[0034] Figure 7 This is a GC-MS detection chart of 2-phenylethanol described in the test example of the present invention.

[0035] Figure 8 A schematic diagram showing the metabolic pathway for the fermented milk of the present invention to form rose fruit aroma. DETAILED DESCRIPTION

[0036] The examples are provided to better illustrate the present invention, but the present invention is not limited to the examples. Therefore, those skilled in the art may make non-essential improvements and adjustments to the implementation scheme according to the above invention content, which still fall within the protection scope of the present invention.

[0037] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0038] The present invention will be described in detail below through examples.

[0039] It should be understood that the following examples are only used to further explain and illustrate the contents of the present invention in detail, but are not used to limit the present invention.

[0040] The Lactococcus lactis used in the following examples include: Lactococcus lactis subsp. cremoris (strain number: CICC 24337) and Lactococcus lactis subsp. lactis (strain number: CICC 6246).

[0041] The following examples relate to the sensory quantitative description analysis (QDA analysis method) process:

[0042] 1. Common reference standards:

[0043] (1) ISO 8586:2012 Sensory analysis. General guidelines for the selection, training and monitoring of selected assessors and professional sensory assessors;

[0044] (2) GB / T 16291.1-2012 Sensory analysis. General guidelines for the selection, training and management of assessors. Part 1: Selection of assessors;

[0045] (3) GB / T 29604-2013 General Guidelines for Establishing Sensory Characteristics Reference Samples for Sensory Analysis;

[0046] (4) GB / T 16860-1997 Sensory analysis method Texture profile test.

[0047] 2. Sensory Evaluator

[0048] (1) Basic taste test: Different concentrations of sour, sweet, bitter, salty, fresh and astringent reference substances are mixed into different gradients of aqueous solution for taste sensitivity test. The accuracy rate is above 80% to pass.

[0049] (2) Basic olfactory test and description ability test: Select aroma substances commonly used in work and mix them into different concentrations for smelling and then describe the aroma substances you smell. The accuracy rate must be above 80% to pass.

[0050] (3) Screening of work experience. Sensory evaluators who have more than two years of work experience in dairy product development and quality control are required to participate in the review of this project.

[0051] (4) Interview: Evaluators who have an interest in the training and experiments of this project, are likely to participate in the project, and have strong communication and language skills.

[0052] (5) Individual performance evaluation. The individual performance of the evaluators was evaluated by fermented milk samples with different sweetness and acidity gradients. The degree of variation in their descriptive evaluation was less than or equal to 15%. Using the above method, 12 evaluators were finally selected to participate in the sensory evaluation of the project.

[0053] 3. Sensory evaluation steps

[0054] (1) Establish sensory characteristics descriptors (established according to literature). Train sensory evaluators to make them familiar with the characteristics of this type of product. First, encourage testers to use any words that they can understand and can explain clearly to other members of the evaluation team to describe the product; then guide testers to check the product and write descriptions of those felt attributes in a blank notebook; the leader of the leading group asks each tester to write down what they have written down and displays it on the blackboard, and uses this method to handle all samples to be evaluated; after two rounds of training, let the testers combine these words in a certain format, and at the same time identify those key sensory experiences. Continue 4 to 5 stages of training to complete the development of the scorecard.

[0055] (2) Determine the reference sample. Based on the established descriptors, the sensory analyst provides some similar reference substances corresponding to the descriptors and provides them to the testers during the training period. The reference substance is a raw material, a product or other substance that can represent a specific sensation and describe the sensory attributes.

[0056] (3) Evaluate the intensity of sensory characteristics (before the sample is sampled, arrange for everyone to smell the reference). Provide reference samples of different intensities of each characteristic to each evaluator for training. Require the evaluators to evaluate in order from strong to weak, familiarize themselves with and memorize the feeling of each characteristic and the corresponding intensity scale. Then take out the reference sample of each characteristic as the test sample. If the evaluator's evaluation is correct at more than 65%, the sensory characteristic intensity of the actual sample can be evaluated. A linear scale is used when evaluating the sample intensity.

[0057] (4) 1 hour before the start of the experiment (provide the descriptive word definitions to the sensory personnel, print out the sensory evaluation form and give each person a copy for sensory testing), take the samples out of the refrigerator and allow them to warm to room temperature. Each fermented milk sample is presented in a transparent, odorless plastic cup. The tasting cups are numbered with 3-digit random numbers. After tasting in a separate tasting room, the tasters score each sample based on various sensory indicators and mark the scoring results on the computer.

[0058] (5) Data processing. The evaluation results of each evaluator are converted into scores and input into the computer. The data results are expressed in the form of spider graphs to show the intensity of sensory characteristics. SPSS statistical analysis software can be used to perform variance analysis to reveal whether there are significant differences between the two products in the tested sensory characteristics.

[0059] Embodiment 1:

[0060] The present invention obtains a compounding ratio that can optimize the flavor product by adjusting the starter ingredients and substrate content, as well as optimizing the process of multiple conditions such as inoculation amount, fermentation temperature, starter ratio, substrate addition amount, and bacterial concentration, and produces flavor substances such as 2-phenylethanol, ethyl acetate, and isopentanol. Through SPME-GC-MS detection combined with flavoromics analysis, 2-phenylethanol (which can impart rose fragrance) was detected in multiple parallel samples of fermented milk. Figure 7 ) and flavor substances with an OAV of 15 to 325, such as ethyl acetate that imparts fruity aroma and isoamyl alcohol that imparts milky aroma.

[0061] Specifically, if Figure 1 As shown in the figure, the process optimization of rose fruit flavor fermented milk: optimize two conditions at a time, such as three gradients of temperature and three gradients of inoculation volume, and ferment nine bottles of fermented milk at a time. The QDA expert group sensory test was conducted on these nine bottles of fermented milk, and the one with the best indicators was selected as the basis for optimizing the following conditions. By analogy, three sets of sensory results were obtained, such as Figures 2 to 4 As shown, the QDA result diagrams of optimizing the five conditions in pairs respectively, finally forming the rose fruit flavored fermented milk product of the present invention.

[0062] This embodiment provides a method for preparing the rose fruit-flavored fermented milk, comprising the following steps:

[0063] (1) Preparation of raw milk: Add water to the milk powder raw material and stir thoroughly to dissolve and hydrate; the hydration condition is hydration at 40°C for 0.5h.

[0064] (2) Add 0.7 g of phenylalanine to 1 L of the hydrated raw milk obtained in step (1), and sterilize the raw milk at a temperature of 95° C. for 5 min.

[0065] (3) Adding the activated Kluyveromyces marxianus ALE-X20 to the sterilized raw milk, the inoculation amount being 4%.

[0066] The specific treatment method includes: inoculating frozen Kluyveromyces marxianus ALE-X20 into YPD enrichment liquid culture medium, culturing in a constant temperature shaker at 30°C for 24 hours, and after 2-3 generations of YPD subculture, taking 5 mL of bacterial culture and placing it in a sterile EP tube for centrifugation, centrifuging at 5000×g for 5 minutes (4°C), discarding the supernatant, adding sterile physiological saline and repeating the above operation, collecting the bacterial cells after centrifugation three times, and adding them into 250 ml of milk for fermentation;

[0067] The composition of the YPD enrichment culture medium is: 20 g of anhydrous glucose, 10 g of yeast powder, 20 g of soy peptone, 1 L of distilled water, and sterilized at 121°C for 15 min.

[0068] (4) The prepared sample is fermented at 35±5°C for 9 hours, or fermented at 40-42°C for 3 hours and then cooled to 35±5°C and continued to ferment for 4 hours; stirring is intermittent during the fermentation period, and then cooled and stored to obtain the rose fruit flavored fermented milk.

[0069] Embodiment 2:

[0070] This embodiment provides a method for preparing rose fruit-flavored fermented milk, comprising the following steps:

[0071] (1) Preparation of raw milk: Add water to the milk powder raw material and stir thoroughly to dissolve and hydrate; the hydration conditions are 35°C-50°C for 0.5-1h.

[0072] (2) Add 0.7 g of phenylalanine to 1 L of the hydrated raw milk obtained in step (1), and sterilize the raw milk at a temperature of 95° C. for 5 min.

[0073] (3) Adding an activated composite probiotic agent consisting of Kluyveromyces marxianus ALE-X20 and Lactococcus lactis subsp. cremoris (strain number: CICC 24337) to the sterilized raw milk, the inoculation amount is 4%, wherein the dosage ratio of Kluyveromyces marxianus ALE-X20 to Lactococcus lactis subsp. cremoris is 1:2.

[0074] Specific treatment methods include:

[0075] For the activation treatment method of Kluyveromyces marxianus ALE-X20, see Example 1.

[0076] The cryopreserved Lactococcus lactis was inoculated into M17 liquid culture medium, cultured at 42°C for 24 hours, subcultured for 2 generations, 10 mL of the culture was placed in a sterile EP tube for centrifugation, the culture was centrifuged at 5000×g for 5 minutes (4°C), the supernatant was discarded, sterile physiological saline was added and the above operation was repeated, the cells were collected after centrifugation three times, and added to 250 ml of milk for fermentation;

[0077] The composition of M17 culture medium is: 5g soy peptone, 5g beef extract powder, 5g lactose, 2.5g meat peptone, 2.5g casein, 2.5g yeast extract powder, 19g sodium glycerophosphate, 0.5g ascorbic acid, 0.25g magnesium sulfate, 1L distilled water, sterilized at 121℃ for 15min.

[0078] (4) The prepared sample is fermented at 30±5°C for 12 to 36 hours, or at 40 to 42°C for 4 to 6 hours, then cooled to 30±5°C and continued to ferment for 12 to 24 hours; stirring is performed intermittently during the fermentation period, and then cooled and stored to obtain the rose fruit flavored fermented milk.

[0079] Comparative Example 1:

[0080] In this comparative example, fermented milk was prepared by referring to the method described in Example 1, except that an equal amount of the Kluyveromyces marxianus ALE-X20 was replaced by Lactococcus lactis subsp. cremoris CICC 24337.

[0081] Test Case

[0082] This test example uses SPME-GC-MS detection and flavoromics analysis to compare the flavor characteristics of the fermented milk products prepared in Examples 1 and 2, Comparative Example 1, and two commercially available fermented milk products. Figure 5 As shown, the five groups of samples are:

[0083] CICC 24337+Massachusetts+AA: the rose fruit flavored fermented milk prepared in Example 2;

[0084] CICC 24337+AA: fermented milk prepared in Comparative Example 1;

[0085] Mashi + AA: the rose fruit flavored fermented milk prepared in Example 1;

[0086] Commercially available rose-flavored fermented milk: Ou Ya Dali Ranch-flavored fermented milk (added with grape pulp and double-petal red rose pulp) (Yunnan Ou Ya Dairy Co., Ltd.);

[0087] Commercially available original fermented milk without added sucrose: flavored fermented milk (original flavor) (Yili Suzhou Dairy Co., Ltd.).

[0088] The PCA results of flavoromics are as follows Figure 6As shown in the figure, the blue arrows represent different flavor compounds, and the four different colored dots represent four different groups of samples. The black circle represents the rose fruit aroma sample prepared in Example 2 (CICC 24337+Marsh yeast+AA), the blue circle represents the rose fruit aroma fermented milk sample prepared in Example 1 (i.e., CICC 24337 group without addition, Marsh yeast+AA), the red circle represents the original fermented milk sample without sucrose added purchased from the market, and the green circle represents the rose flavor fermented milk sample purchased from the market. From the analysis of the figure, it can be obtained that the quadrant where the black circle is located has the most blue arrows pointing to it, which means that it contains the most flavor substances. Among them, Phenylethyl Alcohol is the representative substance of rose fragrance 2-phenylethanol. The larger the absolute value of the projection of the arrow on the horizontal axis, the more content of the compound. As shown in the following table, the commercially available rose flavor fermented milk contains less 2-phenylethanol and more cream aroma compounds such as 2-nonanone (2-nonanone), nonanal (nonanal), 2-undecanone (2-undecanone), etc.

[0089]

[0090] Finally, it should be noted that the above is only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred arrangement scheme, a person skilled in the art should understand that the technical solution of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solution of the present invention.

Claims

1. A Kluyveromyces marxianus, characterized in that The Kluyveromyces marxianus is named ALE-X20. The strain is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, classified and named Kluyveromyces marxianus, with a deposit number of CGMCC NO.32221 and a deposit date of October 15, 2024.

2. The Kluyveromyces marxianus according to claim 1, characterized in that The Kluyveromyces marxianus has a good growth and metabolism condition under the acidic condition of pH 4.0-6.

0.

3. A bacterial agent, characterized in that: The method comprises the Kluyveromyces marxianus according to claim 1 or 2.

4. A composite probiotic agent, characterized in that: The composite probiotic agent comprises Kluyveromyces marxianus and Lactococcus lactis as claimed in claim 1 or 2; The lactococcus lactis includes Lactococcus lactis subsp. cremoris and Lactococcus lactis subsp. lactis.

5. The composite probiotic agent according to claim 4, characterized in that: The two strains of bacteria are mixed in a ratio of 1:10 to 1:30 in terms of live bacteria content.

6. A product prepared from the Kluyveromyces marxianus according to claim 1 or 2, or the bacterial agent according to claim 3, or the composite probiotic bacterial agent according to claim 4 or 5, characterized in that: The product is any one of food, health care product and medicine.

7. Use of the Kluyveromyces marxianus as claimed in claim 1 or 2, or the bacterial agent as claimed in claim 3, or the composite probiotic bacterial agent as claimed in claim 4 or 5 in the preparation of a rose-flavored fermented milk product.

8. The use according to claim 7, characterized in that: The application is to increase the content of characteristic flavor substances including 2-phenylethanol, ethyl acetate and isopentanol during the fermentation process, thereby giving the fermented milk product a rose fragrance.

9. A method for preparing rose fruit-flavored fermented milk, characterized in that: The preparation method comprises the following steps: (1) Preparation of raw milk: Add water to the raw milk powder and stir thoroughly to dissolve and hydrate; the hydration conditions are hydration at 35°C-50°C for 0.5-1h, or hydration below 6°C for 8-12h; (2) adding amino acids to the hydrated raw milk obtained in step (1), and sterilizing the raw milk at a temperature of 85° C. to 95° C. for 5 to 15 minutes; Among them, the amount of amino acid added is 1 to 5 g / 1L raw milk; (3) adding the activated Kluyveromyces marxianus according to claim 1 or 2, or the composite probiotic agent according to claim 4 or 5, to the sterilized raw milk, with an inoculation amount of 1% to 10%; (4) The prepared sample is fermented at 30±5°C for 12 to 36 hours, or at 40 to 42°C for 4 to 6 hours, then cooled to 30±5°C and continued to ferment for 12 to 24 hours; stirring is performed intermittently during the fermentation period, and then cooled and stored to obtain the rose fruit flavored fermented milk.

10. The method for preparing rose-flavored fermented milk according to claim 9, characterized in that: In step (2), the amino acid is phenylalanine.

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