Use of k. marxianus ale-x20 in the preparation of milk beer

Abstract

The application provides application of Kluyveromyces marxianus ALE-X20 in preparation of milk beer, the Kluyveromyces marxianus ALE-X20 is preserved in the China General Microbiological Culture Collection Center on October 15, 2024, and the preservation number is CGMCC NO.32221. The application domesticates a parent strain Km-P through adaptive laboratory evolution technology, and obtains an evolution strain ALE-X20 with significantly improved lactic acid tolerance. Experiments prove that the strain can grow normally in a 20g / L lactic acid environment, and the phenethyl alcohol yield is 28.2 times that of the parent strain. The strain is applied to milk beer fermentation, so that the number of volatile aroma compounds in the milk beer is increased to 52, the total concentration is increased by 28.18% compared with the parent strain fermentation product, the concentration of alcohol and ester compounds is increased by 33.87% and 32.43% respectively, and the odor activity value of a plurality of characteristic aroma compounds is significantly improved. The application of ALE-X20 in the preparation of milk beer can get rid of the dependence on exogenous essence, realize the production of milk beer with a clean label, and improve the flavor quality and market competitiveness of milk beer.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, and specifically relates to the application of Kluyveromyces martensii ALE-X20 in the preparation of milk beer. Background Technology

[0002] Milk beer is a popular fermented dairy beverage that combines the nutrition of dairy products with the foam characteristics of beer. It is usually made from whole or skim milk powder, whey, etc., through secondary fermentation by lactic acid bacteria and yeast. High-quality milk beer has a balanced sweet and sour taste, a delicate and long-lasting foam, and multiple flavors of milk, fruit and malt aromas, making it very popular among young consumers.

[0003] Kluyveromyces marxianus (K. marxianus) is an unconventional yeast capable of metabolizing lactose and is widely used in the fermentation of traditional dairy products such as kefir, yogurt, and cheese. Studies have shown that this strain can improve the quality of fermented dairy products by reducing ethanol content, enriching aroma profiles, and increasing acidity. Currently, some dairy beer producers are experimenting with using milk-derived Kluyveromyces marxianus for dairy beer fermentation to replace or partially replace traditional brewer's yeast or brewing yeast.

[0004] However, the following problems still exist in the existing technology: First, in the co-fermentation system of lactic acid bacteria and yeast in milk beer, lactic acid bacteria continuously accumulate lactic acid and other organic acids during fermentation, leading to a rapid drop in the system's pH. Most Max Kluyveromyces strains have limited acid tolerance, and their growth is significantly inhibited in low pH environments (especially below pH 4.0), resulting in decreased metabolic activity and insufficient aroma production. Therefore, traditional milk beer fermentation time is typically controlled at 10-16 hours, during which time yeast aroma production has not yet reached its peak, resulting in a low variety and concentration of aroma compounds in the produced milk beer. To compensate for insufficient aroma, companies commonly add food flavorings, which contradicts current consumer demands for "clean labels" and natural, healthy foods.

[0005] Second, Adaptive Laboratory Evolution (ALE) has been proven to effectively enhance the synthesis capacity of target metabolites and stress tolerance in microorganisms, and it is simple to operate and requires no complex genetic background knowledge. Current research has shown that ALE technology can improve the acid tolerance of brewer's yeast and lactic acid bacteria, but there are no reports of applying ALE technology to Kluyveromyces martensii to enhance its lactic acid tolerance and aroma production in dairy beer co-fermentation systems.

[0006] Third, although the applicant previously obtained a strain named ALE-X20 (accession number CGMCC NO.32221) from Kluyveromyces martensii, which has undergone adaptive evolution, and disclosed the application of ALE-X20 in the preparation of rose-flavored fermented milk (CN119823883A), this disclosure mainly concerns the application of this strain in traditional fermented milk (yogurt). The fermentation system of milk beer differs significantly from that of ordinary fermented milk: milk beer requires secondary fermentation by lactic acid bacteria and yeast, resulting in a final product with a carbonated taste, foam, and a lower pH (typically 3.5-4.8), and different requirements for the composition of aroma substances such as esters and higher alcohols. Whether this strain can adapt to the unique high-acid, high-ethanol environment of milk beer and stably and efficiently produce aroma in this system remains unclear, and the existing technology does not provide any technical guidance.

[0007] Therefore, developing a strain of Kluwer Maxim. that is highly acid-resistant and can still efficiently metabolize and produce rich floral and fruity aroma substances in the later stages of co-fermentation of milk beer, and applying it to the preparation of milk beer, in order to get rid of dependence on exogenous flavorings, achieve clean labeling and natural flavor enhancement, is a technical problem that urgently needs to be solved in this field. Summary of the Invention

[0008] To address the problems existing in the prior art, the purpose of this invention is to provide the application of Max Kluyveromycin ALE-X20 in the preparation of milk beer.

[0009] The objective of this invention is achieved through the following technical solution: The first aspect of this invention provides the application of Kluyveromyces marxianus ALE-X20 in the preparation of milk beer. The Kluyveromyces marxianus ALE-X20 was deposited at the China General Microbiological Culture Collection Center on October 15, 2024, with the accession number CGMCC NO. 32221.

[0010] Furthermore, the Max Kluyveromycin ALE-X20 can improve the lactic acid fermentation resistance of milk beer, while enhancing the rose and fruit aromas of the milk beer.

[0011] Furthermore, the application is as follows: in a co-fermentation system of lactic acid bacteria and yeast, the Kluyveromyces martensii ALE-X20 is able to withstand a high lactic acid environment, thereby extending the fermentation time and increasing the types and contents of aroma compounds.

[0012] A second aspect of the present invention provides a method for preparing milk beer, comprising the following steps: (1) Preparation of fermented milk: Lactic acid bacteria are inoculated into raw milk and fermented to obtain fermented milk; (2) Preparation of acid emulsion: Mix fermented milk with ingredients and water, homogenize and set aside; (3) Milk beer fermentation: The Kluyveromyces ALE-X20 was inoculated into the ortho-acid emulsion prepared in step (2) and fermented at a constant temperature to obtain milk beer.

[0013] Furthermore, in step (1), the lactic acid bacteria are selected from one or more of Streptococcus thermophilus, Lactobacillus bulgaricus, Lactococcus lactis subsp. milk, and Lactococcus lactis subsp. milk.

[0014] Furthermore, in step (2), the ingredients include L-phenylalanine.

[0015] Furthermore, in step (3), the inoculation concentration of Kluyveromyces martensii ALE-X20 is... ~ CFU / mL; fermentation temperature 28-37℃, fermentation time 24-72 hours.

[0016] A third aspect of the present invention provides a milk beer prepared by the method described in the second aspect.

[0017] A fourth aspect of the present invention provides a compound fermentation agent for preparing milk beer, the compound fermentation agent comprising: Kluyveromyces macrocephala ALE-X20 and lactic acid bacteria.

[0018] Furthermore, the lactic acid bacteria are selected from one or more of Streptococcus thermophilus, Lactobacillus bulgaricus, Lactococcus lactis subsp. milk fat, and Lactococcus lactis subsp. milk.

[0019] Information on strain preservation: The strain of *Kluyveromyces marxianus* has been named ALE-X20 and is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. Its classification name is *Kluyveromyces marxianus*, with accession number CGMCC NO. 32221, and the deposit date is October 15, 2024.

[0020] The advantages of this invention compared to the prior art are as follows: 1. The *Kluyveromyces martensii* ALE-X20 obtained through adaptive laboratory evolution (ALE) in this invention exhibits excellent growth ability in a high-stress environment containing 20 g / L lactic acid. Comparative experiments show that under 20 g / L lactic acid stress, the growth of the parental strain Km-P was completely inhibited, while ALE-X20 was able to maintain normal growth; at the end of the culture, ALE-X20... The value is about 16 times higher than that of the parent strain. ALE-X20 has good adaptability to acidic environments of pH 3.5-4.8 and can maintain an active metabolic state in the later stage of milk beer fermentation (48-72 hours), breaking through the technical bottleneck of short fermentation time and insufficient aroma production caused by insufficient acid resistance of traditional Max Kluwer yeast; 2. Applying the Kluwer Max Marx yeast ALE-X20 described in this invention to milk beer fermentation can significantly increase the content of key aroma compounds in milk beer, especially imparting a rich natural rose and fruit aroma. At the end of fermentation, the total volatile organic compound (VOCs) concentration reached 12,228.81 μg / L, which was 28.18% higher than that of the parent strain Km-P fermentation group. Among them, the increase in the content of characteristic aroma compounds was particularly significant: the yield of 2-phenylethanol, a representative substance of rose aroma, increased by 25.93%, with an aroma activity value of over 100,000; the content of fruit aroma representative substances such as ethyl butyrate and isoamyl acetate increased by 69% and 71% respectively, and the total alcohol and total ester increased by an average of over 30%. The above results indicate that ALE-X20 can significantly enrich the aroma layers of milk beer and form a unique rose and fruit aroma style. 3. The milk beer prepared according to the present invention does not require the addition of edible flavorings. It can produce a rich and natural rose and fruit aroma simply through strain metabolism and amino acid substrates. The sensory score is significantly higher than that of the parent strain group, and the aroma complexity is better than some commercially available products. This provides consumers with a more natural and healthier choice, which is in line with the current development trend of "clean label" in the food industry. Attached Figure Description

[0021] The present invention will be further described below with reference to the accompanying drawings and embodiments: Figure 1 This illustrates the laboratory evolutionary process of adaptation of the parent strain Km-P; Figure 2 The effect of ALE on the growth and phenylethanol production capacity of K. marxianus under different lactic acid concentrations is shown; where (ab): growth of Km-P and ALE-X20 at lactic acid concentrations of 0 g / L and 20 g / L; (cd): phenylethanol production by Km-P and ALE-X20 at lactic acid concentrations of 0 g / L and 20 g / L; ns indicates no significant difference. p < 0.05, p < 0.01, p < 0.001, p < 0.0001; Figure 3 , Figure 4 , Figure 5 Comparative genomic analysis of Km-P and ALE-X20 is shown; among them, Figure 3 (a) Single nucleotide polymorphism (SNP) analysis; Figure 3 For (b) Insertion and Deletion (Indel) analysis; Figure 4 Gene Ontology (GO) enrichment analysis for mutated genes; Figure 5 Enrichment analysis of mutant gene pathways for the Kyoto Encyclopedia of Genes and Genomes (KEGG); Figures 6-9 The differences in VOCs between Km-P and ALE-X20 during the fermentation of milk beer are shown; among them, Figure 6 The classification and total concentration of VOCs in Km-P and ALE-X20 are shown; Figure 7 , Figure 8 Two heatmaps show the relative abundance of VOCs in Km-P and ALE-X20; Figure 9 (a) is the PCA score chart; Figure 9 (b) is the PCA load diagram; Figures 10-13 The PLS-DA analysis results for Km-P and ALE-X20 are shown; among them, Figure 10 The PLS-DA score chart is shown on the right, with the legend representing different times and strains. Figure 11 PLS-DA load diagram; Figure 12 VIP results; Figure 13 For the substitution test; Figure 14 The QDA sensory evaluation results of the milk beer samples are shown; (a) radar charts of different milk beer samples; (b) PCA score charts; where P and ALE represent Km-P and ALE-X20, respectively, and 10 and 72 represent fermentation time (h); three commercial milk beers, TR (Tianrun Milk Beer), XYC (Xiyuchun Milk Beer) and LDR (Landai Rose Milk Beer), are used as references. Detailed Implementation

[0022] The embodiments described are provided to better illustrate the present invention, but are not intended to limit the scope of the invention to the embodiments described. Therefore, non-essential improvements and adjustments made to the embodiments by those skilled in the art based on the above description are still within the scope of protection of the present invention.

[0023] The endpoints and any values ​​of the ranges disclosed herein 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 the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0024] The present invention will be described in detail below through embodiments. It should be understood that the following embodiments are only used to exemplify and further explain and illustrate the content of the present invention, and are not intended to limit the present invention. In the embodiments, the Kluyveromyces marxianus is named Km-P, purchased from the China Industrial Microbial Culture Collection Center (CICC), address: Building 6, No. 24, Jiuxianqiao Middle Road, Chaoyang District, Beijing, China National Research Institute of Food Fermentation Industries, postcode: 100015; classified as Kluyveromyces marxianus; accession number: CICC NO.1953, accession date: May 23, 2007.

[0025] Example 1: Obtaining and identifying Kluyveromyces martensii ALE-X20 This embodiment provides a method for obtaining Kluyveromyces martensii ALE-X20, which is lactic acid resistant and has high aroma-producing capacity.

[0026] 1. Parental strain The parent strain was Kluyveromyces marxianus Km-P, which was deposited at the China Industrial Microbial Culture Collection Center (CICC) with accession number CICC NO.1953.

[0027] 2. Activation and purification of parental strains The Km-P strain, cryopreserved in glycerol tubes and stored at -80℃, was purified by streak plating on YPD solid medium (10 g / L yeast extract, 20 g / L peptone, 20 g / L glucose, 20 g / L agar). After incubation at 30℃ for 24–48 hours, single colonies with regular morphology and no overlap with other colonies were selected. These single colonies were then amplified in YEPD liquid medium (10 g / L yeast extract, 20 g / L peptone, 20 g / L glucose) at 30℃ and 200 r / min to the logarithmic growth phase, yielding a seed culture.

[0028] 3. Adaptive Laboratory Evolution (ALE) Adaptive evolution experiments were conducted using an EVOL Cell (Luoyang Huaqing Tianmu Biotechnology Co., Ltd.). This device can continuously monitor microbial growth (…). The strain was allowed to ferment for several days under certain conditions, and the reactor environment was flexibly adjusted using oxygen partial pressure and temperature control technology.

[0029] Before evolution, the parental strain Km-P was activated to the late logarithmic growth phase in a modified fermentation medium (20 g / L glucose, 5 g / L yeast extract, 0.4 g / L magnesium sulfate heptahydrate, 2 g / L potassium dihydrogen phosphate, and 5 g / L phenylalanine), and then inoculated into the reactor at a 2% (v / v) inoculum. The reactor was connected to two culture medium storage bottles via piping: one containing 200 mL of lactate-free fermentation medium, and the other containing 200 mL of fermentation medium containing 20 g / L lactate.

[0030] The ALE experimental parameters were set as follows: culture temperature 36℃, oxygen concentration 10%, detection wavelength 600 nm, inoculum size 2% (v / v), lactate concentration gradient 0-20 g / L, subculture interval 20 hours, and detection interval 2 hours. After setting the parameters, the ALE experiment was started. When Km-P showed stable growth, the culture medium was collected using a sterile syringe and stored at -80℃. The obtained evolutionary strain was named ALE-X20.

[0031] 4. Analysis of ALE process results like Figure 1 As shown, during the 600-hour adaptive evolution process of the parental strain Km-P, the lactic acid concentration in the culture medium gradually increased from 0 g / L to 20 g / L. In the initial stage of the experiment, the bacterial cells... A brief decrease occurred, followed by a gradual recovery. During the first 150 hours of ALE, Km-P maintained normal growth when lactate concentration was between 0-12 g / L. However, when lactate concentration rose to 14-18 g / L, mild growth inhibition occurred, manifested as… A moderate decrease and fluctuation in growth status were observed. Significant growth inhibition was observed when the lactate concentration increased to 19 g / L. The value dropped from 7.81 to 4.90, a decrease of 37.26%. Nevertheless, after another 180 hours of enrichment culture... The value then rebounded from 4.90 to 6.28. This recovery in growth indicates that Km-P has gradually adapted to a high lactic acid environment and acquired tolerance to lactic acid concentrations up to 20 g / L. The evolved strain ALE-X20 was successfully screened through this ALE process.

[0032] 5. Preservation information for ALE-X20 The Kluyveromyces marxianus ALE-X20 obtained in this embodiment was deposited on October 15, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.32221 and classified as Kluyveromyces marxianus.

[0033] Example 2: Phenotypic Validation of Maskylvae yeast ALE-X20 This embodiment compares and evaluates the growth performance and aroma production capacity of the parent strain Km-P and the evolved strain ALE-X20.

[0034] After activation, each strain was inoculated into fermentation media without lactic acid and with 20 g / L lactic acid, respectively, and cultured at 30℃ with shaking at 200 rpm until the stationary phase. Samples were taken every 2 hours during the culture process for analysis. The values ​​were used to monitor cell growth. Simultaneously, fermentation broth was collected at 18, 24, 30, and 36 hours of culture to determine the yield of phenylethanol.

[0035] The results are as follows Figure 2 As shown. In lactate-free medium, Km-P and ALE-X20 showed similar overall growth trends, with ALE-X20 growing slightly better than Km-P. However, under 20 g / L lactate stress, Km-P showed complete growth inhibition, while ALE-X20 maintained normal growth. At the end of the culture, ALE-X20... It is about 16 times higher than Km-P.

[0036] Regarding phenylethanol production, there was no statistically significant difference between the two strains in lactate-free medium (except at 18 and 36 hours, p < 0.05). However, under 20 g / L lactate stress, ALE-X20 showed a significantly higher phenylethanol production than Km-P throughout the fermentation process (p < 0.0001). At the end of the culture, the phenylethanol concentration of ALE-X20 was 28.2 times that of Km-P. These results indicate that ALE-X20 has enhanced cell growth and aroma compound production capabilities under lactate stress.

[0037] Example 3: Comparative genomics analysis of Kluyveromyces martensii ALE-X20 In this embodiment, the genomes of Km-P and ALE-X20 were resequencing to reveal genetic variation characteristics.

[0038] The *K. marxianus* DMKU3-1042 genome (https: / / www.ncbi.nlm.nih.gov / datasets / genome / GCF_001417885.1 / ) was used as a reference genome. Cell pellets containing Km-P and ALE-X20 were sent to Shanghai Meiji Biopharmaceutical Technology Co., Ltd. for DNA extraction, library construction, sequencing, genome assembly, and annotation. Sequencing was performed in... The PE150 strategy was used on the platform. After quality control filtering, the raw data was aligned to a reference genome using BWA-MEM and processed according to the GATK Best Practices workflow. SnpEff software was used to perform functional annotation of variants in conjunction with gene prediction information from the reference genome, followed by GO enrichment analysis and KEGG pathway enrichment analysis.

[0039] The results are as follows Figures 3-5 As shown in Table 1. Comparative genomic analysis predicted 1551 protein-coding genes, of which 1210 were functionally annotated, and SNP and InDel analyses were performed. During ALE, genomic variation in evolutionary strains can arise through various mechanisms, including the accumulation of point mutations and structural variations resulting from insertions, deletions, horizontal gene transfer, and recombination-dependent intragenomic rearrangements. SNPs represent the main forms of genetic variation in microbial genomes. A total of 8368 SNPs were identified ( Figure 3 Of the 4367 SNPs identified (a), 1208 were nonsynonymous mutations and 965 were synonymous mutations. Among the nonsynonymous mutations, 1188 were missense mutations, the most common type, which can alter protein structure and potentially affect its function. Furthermore, 2504 SNPs were located in upstream regulatory regions of the gene, and 1899 were located in downstream regions. Figure 3 (b) 123 frameshift mutations and 144 non-frameshift mutations were identified in the protein-coding region. In the non-coding region, 1565 insertions and deletions were located upstream of the gene, and 1045 were located downstream. The high proportion of SNPs and insertions / deletions in the upstream region suggests that these mutations may have a significant impact on transcriptional regulation and gene expression levels, potentially affecting cellular function and phenotypic traits.

[0040]

[0041] Comparative genomic analysis based on Km-P and ALE-X20 identified 10 potential candidate genes associated with enhanced acid tolerance and another 10 candidate genes associated with enhanced aroma compound biosynthesis (Table 1). Acid tolerance-related genes were divided into four main functional groups: antioxidant responses and reactive oxygen species (ROS) scavenging, membrane stability maintenance, proton pumps and pH homeostasis, and the trehalose metabolic pathway. Under lactic acid stress, ROS accumulation disrupts cellular homeostasis. CTA1, YBP1, and POS5 work synergistically to mitigate oxidative stress. POS5 encodes mitochondrial NADH kinase, which provides NADPH for the glutathione and thioredoxin antioxidant system. YBP1 acts as an oxidative stress sensor. Exposure to… Subsequently, it forms a ternary complex with transcription factor Yap1 and peroxidase Gpx3, promoting the oxidation and nuclear translocation of Yap1, thereby upregulating the expression of antioxidant genes. CTA1 encodes catalase, which directly... It is converted into water and oxygen. Membrane lipids and sterols are crucial for maintaining the barrier properties of the plasma membrane under stress conditions. IPT1 regulates sphingolipid composition, affecting membrane fluidity, proton permeability, and overall membrane integrity. ERG2 and ERG7 are key components of the sterol biosynthetic pathway, affecting the sterol skeletal structure, leading to reduced membrane fluidity and limiting the passive diffusion of weak acids. Low pH and weak acid stress lead to proton accumulation, disrupting membrane potential and inhibiting cell growth. VMA16, along with Vma3p and Vma11p, forms proton channels, utilizing ATP hydrolysis to convert protons into water and oxygen. Actively transported into the vacuoles, thereby reducing intracellular acid load and maintaining the membrane potential required for secondary ion transport, PDR12 is an acid-responsive ABC transporter. Its expression is significantly upregulated under weak acid or low pH stress, promoting the efflux of weak organic acids into the extracellular environment. The trehalose metabolic pathway plays a crucial role in stress resistance by acting as a carbon reserve and osmotic protectant. TSL1 is involved in trehalose biosynthesis, while TRE2 is involved in trehalose degradation, providing carbon sources and ATP to support growth recovery under stress conditions.

[0042] To further elucidate the biological functions / processes affected by these mutations in the evolved strains, we performed enrichment analysis, which revealed significant enrichment of several key metabolic pathways. Figure 4The study revealed GO items (p < 0.05) that significantly enriched the mutant gene in the molecular function (MF), cellular component (CC), and biological process (BP) categories. In the BP category, the mutant gene was primarily involved in carbohydrate metabolism and biosynthesis, lipid metabolism and biosynthesis, energy metabolism, nucleic acid metabolism, amino acid metabolism, and the biosynthesis of aromatic compounds. For MF, the mutant gene was mainly involved in DNA binding, enzyme activity, transcriptional regulation, ADP binding, and cofactor binding. CC-related mutant genes were mainly located in the extracellular region, early budding stage, enzyme complexes, membrane protein complexes, cell membrane, and vesicles. GO enrichment analysis showed that transferase activity (transfer of phosphate-containing groups), DNA binding, and aromatic compound biosynthesis were the most significantly enriched items. In evolved strains, specific transferases may need to adjust their activity to meet new metabolic demands or compensate for defects in metabolic pathways. Furthermore, gene mutations also affect the structure and function of DNA. DNA-binding proteins, such as transcription factors and other regulatory proteins, can recognize these changes and respond by regulating the expression of mutant genes to maintain genome stability. Aromatic compounds mainly originate from the central metabolic pathways of the three standard aromatic amino acids: tyrosine, phenylalanine, and tryptophan. Related genes include ARO8, ARO9, ADHs, PDC, LEU3, LEU5, and ILV2. (KEGG results) Figure 5 The results showed that the mutant gene was significantly enriched in the biosynthesis of secondary metabolites, carbon metabolism, and oxidative phosphorylation pathways. In yeast, secondary metabolites typically include high-value compounds such as fatty acids and their esters, higher alcohols, and terpenoids, which are key contributors to aroma characteristics. Carbon metabolism plays a central role in supplying energy and precursor compounds by breaking down or converting carbon sources through glycolysis, the tricarboxylic acid (TCA) cycle, and oxidative phosphorylation. The main function of oxidative phosphorylation is to convert energy from the electron transport chain into AP, thereby providing the chemical energy required for yeast growth and development. Enrichment analysis results indicate that ALE-X20 may enhance its tolerance to lactic acid stress and improve aroma production efficiency by regulating key metabolic pathways and gene expression, thereby maintaining genome stability and supporting normal cell growth.

[0043] Example 4: Preparation of milk beer using Kluyveromycin ALE-X20 This embodiment provides a specific method for preparing milk beer using the Max Kluyveromycin ALE-X20 described in this invention.

[0044] 1. Preparation of Fermented Milk Reconstituted milk was prepared by dissolving 11.5% (w / w) whole milk powder (Fonterra Group Ltd., Beijing) and 6.5% (w / w) sucrose (Guangzhou Fuzheng Donghai Food Co., Ltd., Beijing) in distilled water at 60°C. The resulting mixture was homogenized using a high-pressure homogenizer (SRH, Shenlu, Shanghai) at 20 MPa, followed by pasteurization at 95°C for 5 minutes and cooling to 37°C. 0.0012 g of the commercial starter Chr. Hansen YoFlex Premium 5.0 (Horsholm, Denmark), containing *Streptococcus thermophilus* and *Lactobacillus delbrueckii* subsp. bulgaricus*, was inoculated into the reconstituted milk and fermented at 37°C until the final pH reached 4.6. The resulting fermented milk was then stored at 4°C for later use.

[0045] 2. Preparation of ortho-acid emulsion The dried ingredients were thoroughly mixed, including 0.4% (w / w) sodium carboxymethyl cellulose (Shandong Zhongyuan Biotechnology Co., Ltd., Shandong), 6.5% (w / w) sucrose, and 0.079% (w / w) food-grade L-phenylalanine (Zhongyan Ingredients Trading Co., Ltd., Henan). Then, 62% (w / w) drinking water was added to dissolve the dry ingredients. The resulting mixture was heated in a 60°C water bath with continuous stirring until all the dry powder was completely dissolved. After cooling to approximately 30°C, 31% (v / v) of the fermented milk obtained in step 1 was added and thoroughly mixed. The final mixture was homogenized at 20 MPa and stored at 4°C for later use.

[0046] 3. Yeast activation and inoculation The *Kluyveromyces martensii* ALE-X20 obtained in Example 1 was removed from its -80°C cryopreservation tube and activated by streaking on YPD solid medium, then incubated at 30°C for 24-48 hours. Single colonies were picked and inoculated into YPD liquid medium, and cultured at 30°C with shaking at 200 rpm for 24 hours until the logarithmic phase. The activated yeast culture was centrifuged at 8000 rpm for 15 minutes to collect the cells, washed twice with PBS buffer (Beijing Jianqiang Weiye Technology Co., Ltd., Beijing), and resuspended in PBS buffer to prepare a yeast suspension. The yeast suspension was then... The inoculation concentration of CFU / mL was introduced into the orthoacid emulsion prepared in step 2.

[0047] 4. Milk beer fermentation The inoculated mixture was fermented in a constant temperature water bath at 30℃ for 72 hours. Samples were taken at 0, 10, 24, 48, and 72 hours during fermentation to determine the pH value of the fermentation broth using a pH meter (Mettler-Toledo, Shanghai). After fermentation, the milk beer samples were cooled to 4℃ for storage.

[0048] Example 5: Analysis of volatile compounds in milk beer This embodiment analyzes the volatile flavor compounds in the milk beer sample prepared in Example 4.

[0049] 1. SPME-Arrow extraction of volatile compounds Take 5 g of milk beer sample and place it in a 20 mL brown headspace vial with a white silicone septum cap. Add 0.5 g NaCl to each sample, followed by 10 μL of the internal standard 2-methyl-3-heptanone (mass concentration 0.0816 mg / mL, Sigma-Aldrich Trading Co., Ltd., Shanghai). Equilibrate the samples in a 45℃ water bath for 30 minutes. After equilibration, extract using a Shimadzu Smart SPME Arrow (DVB / C-WR / PDMS, 20 mm × 1.1 mm outer diameter, df = 120 μm) at 45℃ for 30 minutes. After extraction, place the fiber in the injection port and desorb at 230℃ for 5 minutes, maintaining the injection port temperature at 250℃.

[0050] 2. GC-MS Analysis The detection of volatile compounds was performed using a GCMS-TQ8050 NX triple quadrupole gas chromatograph-mass spectrometer (GC-MS / MS) equipped with an SH-PolarWax capillary column (60 m × 0.25 mm id, df = 0.25 μm). Helium was used as the carrier gas at a flow rate of 1.0 mL / min. Splitless injection was used, and the injection port temperature was set to 250 °C. The chromatographic separation program was as follows: initial temperature of 40 °C held for 5 minutes, followed by a temperature ramp to 200 °C at 3 °C / min and a hold at 200 °C for 5 minutes. Mass spectrometry detection was performed in electron impact ionization (EI) mode with an ionization energy of 70 eV. The ion source temperature and interface temperature were set to 200 °C and 250 °C, respectively. The mass spectrometry acquisition mode was full scan, with a scan mass range of 35–600 m / z.

[0051] 3. Qualitative and quantitative analysis Qualitative analysis of volatile compounds in milk beer was performed using mass spectrometry characteristics compared to the NIST20 mass spectrometry database and retention indices (RIs). Quantitative analysis was conducted using the internal standard method. The odor activity value (OAV) of volatile aroma compounds was calculated as the ratio of their concentration in the sample to their odor threshold in water. Generally, an OAV ≥ 1 indicates that the compound can be perceived by the human nose, and the higher the OAV, the greater its odor intensity and contribution to the overall aroma.

[0052] 4. Results A total of 53 VOCs were identified during the fermentation of milk beer. Km-P and ALE-X20 contained 44 and 52 VOCs, respectively. These VOCs were classified into seven types: alcohols, aldehydes, acids, esters, ketones, and terpenes. The total concentration of VOCs in Km-P and ALE-X20 increased over time ( Figure 6 In the initial stage (0h), yeast activity had not yet begun, resulting in a relatively low total VOC concentration compared to later fermentation stages. VOCs were predominantly acids and ketones. In subsequent fermentation stages, alcohols became the dominant volatile organic compounds, followed by esters and acids. Ester concentrations increased rapidly at 48h and 72h, likely due to environmental factors such as decreased pH and fluctuations in protein levels in the culture medium. In the early to mid-stages of milk-beer fermentation (10h and 24h), the total VOC concentration of Km-P was higher than that of ALE-X20. However, as fermentation progressed, lactic acid stress likely inhibited the metabolic activity of Km-P, resulting in a final VOC concentration of 9539.99 μg / L at the end of fermentation (72h). In contrast, ALE-X20 exhibited enhanced metabolic activity in the later stages of fermentation, reaching 12,228.81 μg / L, a 28.18% increase over Km-P. The results are as follows... Figures 6-8 As shown. Furthermore, the average alcohol concentration in ALE-X20 (6899.19 μg / L) was 33.87% higher than that in Km-P (5153.75 μg / L); the average ester concentration in ALE-X20 (4444.49 μg / L) was 32.43% higher than that in Km-P (3356.12 μg / L). Notably, several volatile aromatic compounds, including tetraalkylaldehyde (citrus, musk, wax), citronellol (floral, wax), ethyl phenylacetate (rose), 1-pentanol (balsam), 1-decyl alcohol (floral), and indole (floral), were identified only in ALE-X20 and were absent in Km-P.

[0053] Principal component analysis (PCA) revealed two principal components (PC1 and PC2), accounting for 59.2% and 11.4% respectively, with a cumulative contribution of 70.6% (R²X = 0.706). (In the PCA scoring plot...) Figure 9 In (a), milk beer samples at different fermentation times showed clear separation along the principal components, indicating that VOCs in milk beer changed with fermentation time. Furthermore, Km-P and ALE-X20 showed distinct grouping patterns, especially in the later stages (48 h and 72 h, pH = 3.77 and 3.71, respectively), suggesting potential metabolic differences between Km-P and ALE-X20 under lactic acid stress. (PCA loading diagram) Figure 9 As shown in b), 2,3-butanedione, ethyl butyrate, and acetoin are located on the negative axis of PC1, while phenylethanol, isoamyl alcohol, and various esters are positioned along the positive axis. This spatial distribution suggests that these compounds contribute to the separation of samples on PC1. Regarding PC2, compounds such as indole, (2R,3R)-2,3-butanediol, 2-nonanone, 1-decanol, dimethyl sulfone, 1-octanol, and 2-heptanone exhibit high negative loadings, while β-ionone, nonanal, and butyl acetate exhibit high positive loadings. Therefore, these compounds are more closely correlated with the changes in capture along PC2, indicating that they contribute to sample separation in this dimension.

[0054] In this study, 25 odor-active compounds (OAV ≥ 1) were identified (see Table 2), including 3 alcohols, 4 aldehydes, 3 acids, 9 esters, and 6 ketones. At the end of fermentation (72 h), the OAVs of phenylethanol (OAV: 86,252–108,632), isoamyl alcohol (OAV: 195–255), phenylacetaldehyde (OAV: 451–991), ethyl acetate (OAV: 205–270), ethyl butyrate (OAV: 5,920–10,020), isoamyl acetate (OAV: 213–366), ethyl hexanoate (OAV: 2,699–3,434), phenylethyl acetate (OAV: 62–73), and 2,3-butanedione (OAV: 151–261) were higher than those of other odor-active compounds, indicating that these odor-active compounds play a dominant role in shaping the aroma characteristics of milk beer. Therefore, the aroma characteristics of milk beer are mainly characterized by rose, fruit, cream, and green aromas. Furthermore, the total OAV of ALE-X20 is higher than that of Km-P, further demonstrating the enhanced aroma-generating ability of ALE-X20 under lactic acid stress. Higher alcohols are important volatile aroma compounds in fermented beverages. At appropriate concentrations, higher alcohols contribute floral and fruity aromas, enhancing the complexity of the aroma and sensory experience. Among the higher alcohols identified in this study, phenethyl alcohol, characterized by a rose aroma, exhibited the highest OAV. Its OAV increased over time in both Km-P and ALE-X20. At 72 h, the concentration of phenethyl alcohol in ALE-X20 was 1,629.49 ± 104.92 μg / L, while the concentration in Km-P was 1,293.79 ± 97 μg / L, an increase of 25.93%. This enhancement can be attributed to the biosynthesis of phenylethanol from phenylalanine via the Ehrlich pathway, which involves key genes such as ARO8, ARO9, ARO10, and AHD. Mutated genes involved in this pathway are listed in Table 1. These findings suggest that ALE-X20 possesses a more efficient phenylethanol biosynthesis pathway, likely due to the upregulation of key gene expression, thereby enhancing the conversion of phenylalanine to phenylethanol.

[0055] Esters impart rich fruity, sweet, and pleasant aromas to fermented beverages. In this study, two major classes of esters were identified in milk beer samples: fatty acid ethyl esters and higher alcohol acetate esters. Among the ethyl esters, ethyl acetate (apple, banana) and ethyl butyrate (pineapple) had relatively high OAVs. At 72 h, the concentrations of ethyl acetate were: 1,351.23 ± 35.66 μg / L in ALE-X20 and 1,028.24 ± 101.5 μg / L in Km-P, an increase of 31.41%; ethyl butyrate was 10.02 ± 0.07 μg / L in ALE-X20 and 5.92 ± 0.25 μg / L in Km-P, an increase of 69.26%. Acetates included isoamyl acetate (sweet, fruity, banana flavor) and phenylethyl acetate (rose, fruity, tropical flavor). The concentrations of these compounds are as follows: isoamyl acetate—54.98 ± 1.76 μg / L in ALE-X20 and 32.07 ± 1.04 μg / L in Km-P, an increase of 71.43%; phenethyl acetate—1,390.1 ± 64.05 μg / L (ALE-X20) and 1,185.02 ± 61.99 μg / L (Km-P), an increase of 17.31%.

[0056]

[0057] Partial least squares discriminant analysis (PLS-DA) is a supervised statistical method that effectively distinguishes and identifies differences between samples and determines characteristic biomarkers. PLS-DA modeling allows for the assessment of predictor importance in projected (VIP) scores, quantifying the specific contribution of individual variables to the aroma characteristics of milk beer. Volatile compounds with VIP > 1 are considered potential characteristic flavor biomarkers. In this study, Km-P and ALE-X20 were compared using PLS-DA based on 17 key odor-active compounds (OAV ≥ 1, p < 0.05). (See the scoring chart). Figure 10 As shown in the figure, PC1 and PC2 account for 71.3% and 14.8% of the total variance, respectively. = 0.92 and = 0.696, indicating the model is robust and reliable. On PC1, groups P-10, P-24, ALE-10, and ALE-24 are in the positive direction, while P-48 and ALE-48 are in the negative direction. On PC2, ALE-72 is in the positive direction, and P-72 is in the negative direction. This distribution indicates that fermentation time and strain type significantly affect the aroma characteristics of milk beer, consistent with previous PCA results. (Permutation test...) Figure 13 The left side shows all the blue... The values ​​are all lower than the origin on the right. The Y-intercept is less than 0.5. When the horizontal coordinate is 1, Less than ,and Very close This indicates that the original model is reliable and there is no overfitting. Figure 12 Eight volatile compounds with VIP > 1 were identified: nonanal, hexanoic acid, phenylacetaldehyde, 2,3-butanedione, isobutyraldehyde, 2-heptanone, phenethyl acetate, and ethanol. (See loading diagram.) Figure 11 The results indicate that, apart from 2,3-butanedione, these compounds are key contributors distinguishing Km-P from ALE-X20. Notably, nonanal, phenylacetaldehyde, isobutyraldehyde, and 2-heptanone, primarily aldehydes, were identified as characteristic compounds of Km-P. Aldehydes typically impart a green and fresh aroma to dairy products.

[0058] Example 6: Sensory evaluation of milk beer This embodiment uses quantitative descriptive analysis (QDA) to perform sensory evaluation on milk beer samples.

[0059] The sensory evaluation panel consisted of 10 trained judges (5 men and 5 women, aged 23-27). A consensus-based aroma descriptor database was established through panel discussion, including seven aroma descriptors: rose, fruit, grass, fermentation, cream, butter, and frankincense. The intensity of each aroma was scored on a continuous scale (0 representing the lowest intensity, and 9 representing the strongest). Reference samples for evaluation included: Km-P milk beer fermented for 10 and 72 hours (P-10 and P-72), ALE-X20 milk beer fermented for 10 and 72 hours (ALE-10 and ALE-72), and three commercially available milk beers (Tianrun Milk Beer TR, Xiyu Chun Milk Beer XYC, and Landai Rose Milk Beer LDR) as controls.

[0060] The results are as follows Figure 14 As shown, both the P-72 and ALE-72 groups exhibited stronger rose and fruit aromas than the 10-hour group. ALE-72 scored an average of 7.80 points for rose aroma, while P-72 scored 6.77 points; their fruit aroma scores were 7.03 and 6.63 points, respectively. The P-72 group showed a stronger green aroma (6.70 points), while ALE-72 scored 5.63 points. Compared to commercially available milk beer, the milk beer produced by this invention exhibits greater aroma complexity and more natural characteristics.

[0061] Comparative Example 1: Preparation of milk beer using parental strain Km-P Milk beer was prepared according to the method of Example 4, except that the Kluyveromyces martensii ALE-X20 was replaced in equal amounts with the parent strain Km-P (CICC NO. 1953). Flavor analysis and sensory evaluation were performed according to the methods of Examples 5 and 6.

[0062] like Figures 6-9 The results showed that the total VOCs concentration in the milk beer sample fermented with Km-P for 72 hours was 9,539.99 μg / L, and the 2-phenylethanol content was 1,293.79 μg / L, both significantly lower than those in the ALE-X20 fermentation group. Sensory evaluation ( Figure 14 In group a), the rose and fruit aroma scores were 6.77 and 6.63 respectively, which were lower than those of the ALE-X20 fermentation group.

[0063] Comparative Example 2: Preparation of milk beer without adding L-phenylalanine Milk beer was prepared according to the method in Example 4, except that L-phenylalanine was not added in step 4.2. The results showed that the yield of 2-phenylethanol was significantly reduced and the intensity of the rose aroma was significantly weakened.

[0064] Finally, it should be noted that the above description is only used to illustrate the technical solutions of the present invention and is not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the present invention.

Claims

1. The application of Kluyveromycin ALE-X20 in the preparation of milk beer, characterized in that, The Kluyveromyces marxianus ALE-X20 was deposited at the China General Microbiological Culture Collection Center on October 15, 2024, with accession number CGMCC NO. 32221.

2. The application according to claim 1, characterized in that, The Max Kluyveromycin ALE-X20 can improve the lactic acid fermentation resistance of milk beer, while enhancing the rose and fruit aromas of milk beer.

3. The application according to claim 1, characterized in that, The application is as follows: in a co-fermentation system of lactic acid bacteria and yeast, the Max Kluyveromyces ALE-X20 is able to withstand a high lactic acid environment, thereby extending the fermentation time and increasing the types and contents of aroma compounds.

4. A method for preparing milk beer, characterized in that, The preparation method includes the following steps: (1) Preparation of fermented milk: Lactic acid bacteria are inoculated into raw milk and fermented to obtain fermented milk; (2) Preparation of acid emulsion: Mix fermented milk with ingredients and water, homogenize and set aside; (3) Milk beer fermentation: The Kluyveromyces ALE-X20 was inoculated into the ortho-acid emulsion prepared in step (2) and fermented at a constant temperature to obtain milk beer.

5. The method for preparing milk beer according to claim 4, characterized in that, In step (1), the lactic acid bacteria are selected from one or more of Streptococcus thermophilus, Lactobacillus bulgaricus, Lactococcus lactis subsp. milk fat, and Lactococcus lactis subsp. milk.

6. The method for preparing milk beer according to claim 4, characterized in that, In step (2), the ingredients include L-phenylalanine.

7. The method for preparing milk beer according to claim 4, characterized in that, In step (3), the inoculation concentration of Kluyveromyces martensii ALE-X20 is: ~ CFU / mL; fermentation temperature 28-37℃, fermentation time 24-72 hours.

8. A type of milk beer, characterized in that, It is prepared by the method described in any one of claims 4 to 7.

9. A compound fermentation agent for preparing milk beer, characterized in that, The compound fermentation agent comprises: Kluyveromyces macrocephala ALE-X20 and lactic acid bacteria.

10. The compound fermentation agent according to claim 9, characterized in that, The lactic acid bacteria are selected from one or more of Streptococcus thermophilus, Lactobacillus bulgaricus, Lactococcus lactis subsp. milk fat, and Lactococcus lactis subsp. milk.

Images