NL2037597A

NL2037597A - Method for screening lactobacillus casei fermentation agent metabolizing sucrose

Info

Publication number: NL2037597A
Application number: NL2037597A
Authority: NL
Inventors: Zhao Yan; Li Yuan; Luo Yang; Luo Jie; Zhang Hao; Li Yixuan; Wang Ran; Fan Ke; Shen Qingwu; Guo Huiyuan; Wang Pengjie; Zhou Hui
Original assignee: Univ Hunan Agricultural
Priority date: 2023-10-17
Filing date: 2024-05-02
Publication date: 2024-06-03
Also published as: CN117074699A; CN117074699B

Abstract

The present disclosure a method for screening lactobacillus casei fermentation agent metabolizing sucrose. The method directionally screens the lactobacillus casei metabolizing sucrose through an amino acid, sequence of sucrose—6—phosphohydrolase, so as to use 5 the lactobacillus casei obtained through screening as a fermentation agent to apply to fermentation of milk products. Therefore, acid production of the lactobacillus casei in the fermented milk is facilitated, a fermentation rate is accelerated, the quality of the fermented milk is improved, and the method has 10 important application significance in screening of new fermentation agents. (+ Fig. l)

Description

METHOD FOR SCREENING LACTOBACILLUS CASEI FERMENTATION AGENT

METABOLIZING SUCROSE

Technical Field

The present disclosure belongs to the technical field of mi- crobiology and genetic engineering, and specifically to a method for screening lactobacillus casei fermentation agent metabolizing sucrose.

Background

As a kind of typical fermented food, fermented milk has been consumed for thousands of years. The concept of "fermented milk" was first introduced by the Greeks in 100 B.C., and today it is consumed all over the world. A number of studies have shown that the fermented milk is high in vitamin and protein content and easy to absorb by intestinal tracts, and also has important physiologi- cal functions of reducing cholesterol and resisting tumor in addi- tion to having a function of regulating intestinal flora, such that fermented dairy products are highly favored by customers. The fermented milk products use cow milk as a main raw material, and are acidic dairy products prepared through lactic acid bacteria fermentation or common fermentation of lactic acid bacteria and yeast. As a milk product, the fermented milk products have been popular in the market for many years due to their unique taste and flavor. Studies have shown that main factors affecting the consum- ers to buy the fermented milk products are quality, price, availa- bility, and brand, of which quality is the most important, such that improving the quality of the fermented milk products is un- doubtedly the most important means for companies to cater to the market.

During preparation of the fermented milk, a base material is prepared by using pure milk or reconstituted whole milk, or su- crose; and fermentation is performed by adding a fermentation agent, and detection of total acid production, viscosity, microbi- al count, and volatile aroma components is performed after certain acidity or pH is reached, so as to finally evaluate the quality of the fermented milk. Lactose and sucrose metabolism is the main sugar source used by fermentation agent strains. The sucrose me- tabolism has four metabolic pathways. A phosphoenolpyruvate- sucrose phosphotransferase system is the predominant sucrose me- tabolism pathway in lactobacillus, and sucrose-6-phosphohydrolase is the most important rate-limiting enzyme, which is mainly used to hydrolyze end non-reducing B-D-fructofuranoside residues in p-

D-fructofuranoside. However, structural characterization of the sucrose-6-phosphohydrolase has been accomplished only in a few species of lactic acid bacteria (mainly including streptococcus mutans, escherichia coli, etc.), tiny changes in an amino acid se- quence of the sucrose-6-phosphohydrolase currently found can change the substrate specificity of the sucrose-6- phosphohydrolase, directly affecting a folding structure of the sucrose-6-phosphohydrolase, and thus causing differences in the metabolism of sucrose by the strains. Meanwhile, the substrate specificity of the sucrose-6-phosphohydrolase evolves with the strains, and significant differences in enzyme activity and pro- teins without enzyme activity are also found in homologs of other sucrose hydrolase.

Therefore, an urgent problem to be solved in the art is to accurately, rapidly and directionally screen a lactobacillus casei metabolizing sucrose to improve a fermentation acid production rate.

Summary

In order to overcome the problems in the related art, the present disclosure provides a method for screening a lactobacillus casei fermentation agent metabolizing sucrose. The method direc- tionally screens the lactobacillus casei metabolizing sucrose through an amino acid sequence of sucrose-6-phosphohydrolase, so as to use the lactobacillus casei obtained through screening as a fermentation agent to apply to fermentation of milk products.

Therefore, acid production of the lactobacillus casei in the fer- mented milk is facilitated, a fermentation rate is accelerated, the quality of the fermented milk is improved, and the method has important application significance in screening of new fermenta- tion agents.

In order to achieve the above purpose, the present invention may adopt the following technical scheme:

The present invention provides a method for screening a lac- tobacillus casei fermentation agent metabolizing sucrose, compris- ing the following steps:

S1, amplification of a sacA gene: extracting genomic DNA of the lactobacillus casei to be test- ed, and obtaining a full-length sequence of the sacA gene of the lactobacillus casei to be tested through amplification;

PCR primers amplifying the sacA gene comprise nucleotide se- quences shown as SEQ ID NO.1l and SEQ ID NO.2; 52, determination of an amino acid sequence of sucrose-6- phosphohydrolase of the lactobacillus casei to be tested: based on the full-length sequence of the sacA gene acquired in 81, determining the amino acid sequence of the sucrose-6- phosphohydrolase of the lactobacillus casei to be tested through software; and 83, comparison of the amino acid sequence of the sucrose-6- phosphohydrolase comparing the amino acid sequence, which is shown as SEQ ID

NO.3, of the sucrose-6-phosphohydrolase of the lactobacillus casei to be tested obtained in S2, and determining that the lactobacil- lus casei to be tested is able to metabolize sucrose.

Further, the method further comprising the following steps:

S4, verification of utilization of the lactobacillus casei to be tested on sucrose:

S41, activating the lactobacillus casei to be tested; and

S42, determining the utilization of the lactobacillus casei to be tested on the sucrose.

Further, extracting the genomic DNA of the lactobacillus ca- sel to be tested in S1 specifically comprises the following steps: inoculating the lactobacillus casei to be tested in a culture medium, culturing same in a stationary manner, when OD600 = 0.8- 1.0, taking bacterial liquid for centrifugation, removing superna- tant, adding a TES solution to the precipitate, so as to resuspend the bacteria, performing centrifugation, removing supernatant, then adding lysozyme to the bacteria to resuspend the bacteria,

performing digestion, then adding RNase to well mix, then adding a protease K solution to well mix, adding a buffer solution GB for oscillation, performing treatment in a water bath, adding anhy- drous ethanol to well mix, performing centrifugation, and pouring waste liquid; adding a buffer solution GD, performing centrifuga- tion, and pouring waste liquid; adding a washing solution PW for washing, performing centrifugation, and drying the residual wash- ing solution in an adsorbing material; and adding a preheated TE buffer solution dropwise, performing centrifugation, collecting samples, and preserving the samples at a low temperature for later use.

Further, in Sl, the sacA gene is amplified by using a PCR technology; an Ex Tag HS enzyme is selected to establish a 25 pL system; and PCR reaction conditions comprise: 95°C, 33min; [95°C, 30 s; 60°C, 30s; and 72°C, 2 min] x 30 cycles; 72°C, 5 min; and 4°C.

Further, in S4, S41 of activating the lactobacillus casei to be tested comprises the following specific steps: inoculating the lactobacillus casei to be tested in an MRS liquid culture medium, and culturing the lactobacillus casei to be tested at 37+1°C for 12h, and performing strain activation.

Further, in $4, S42 of determining the utilization of the lactobacillus casei to be tested on the sucrose comprises the fol- lowing specific steps: first, determining whether the lactobacillus casei to be tested is able to utilize the sucrose; inoculating the lactobacil- lus casei to be tested in an MRS liquid culture medium, and cul- turing the lactobacillus casei to be tested at 37+1°C for 12h, and detecting sucrose metabolism by the strain; then using the strain that is determined to be able to uti- lize the sucrose as a fermented milk base of a fermentation agent, and calculating a sucrose utilization rate; preparation of a fermented milk: mixing raw milk and the su- crose, performing homogenization and pasteurization, and preparing a fermentation base material after cooling; and inoculating the lactobacillus casei to be tested activated in the fermentation base material according to an addition of 5x10° CFU/g, and perform-

ing culture at 37 °C to obtain the fermented milk; and detection of sucrose content: accurately weighing a sucrose standard to a 100mL volumetric flask, making up to 100 mL with ul- trapure water, wherein mass concentrations of the sucrose respec- 5 tively are 3, 2.5, 2, 1.75, 1.50, 1.25, 1.0, 0.75, 0.50 and 0.25 mg/mL; plotting a standard curve by using the concentration of the standard as a horizontal coordinate and using a peak area as a longitudinal coordinate; weighing the fermented milk to a beaker, and adding deionized water for full dissolving with a magnetic stirrer; adding a Carrez reagent A and a Carrez reagent B in se- quence, and obtaining a mixture after full dissolving; centrifug- ing the mixture, and taking supernatant; diluting the supernatant with distilled water, and performing filtration by using a filter membrane; in combination with a chromatographic column and a dif- ferential Refractive Index Detector (RID), using a sulfuric acid aqueous solution as a mobile phase, and detecting the sucrose con- tent in a fermented milk sample by using an external standard method, so as to calculate the sucrose utilization rate.

The present disclosure has the following beneficial effects:

Existing technologies are short of methods for directionally screening the lactobacillus casei metabolizing sucrose by using amino acid sequencing means. Activated strains are still used and inoculated in a fermentation base material containing sucrose; and after a certain period of incubation, the utilization of the lac- tobacillus casei on the sucrose is determined by determining su- crose content in the base material and calculating a utilization rate of the sucrose, such that the methods are tedious in opera- tion and low in time consumption, and are limited and affected by a detection limit of an experimental equipment, operation tech- niques of testing personnel, making it difficult to guarantee the accuracy of the test. In the method of the present disclosure, the lactobacillus casei metabolizing sucrose is directionally screened through an amino acid sequence of sucrose-é-phosphohydrolase, such that the screening efficiency and accuracy of the lactobacillus casei fermentation agent are greatly improved, and the method has important application significance in screening of new fermenta- tion agents.

Brief Description of the Drawings

The present invention is further explained below in combina- tion with the accompanying drawings and the embodiments.

Fig. 1 is a comparison diagram of utilization of lactobacil- lus casei on sucrose in different groups.

Fig. 2 a comparison diagram of amino acid sequences of su- crose-6-phosphohydrolase SacA of lactobacillus casei strains in different groups.

Fig. 3 is a comparison diagram of sucrose content in ferment- ed milks in different groups.

Detailed Description of the Embodiments

The implementation of the invention is not limited to the following embodiments, and any form of adaptations and / or chang- es made to the invention will fall into the scope of protection of the invention. In the present invention, all the equipment and raw materials can be purchased from the market or commonly used in the industry. Unless otherwise specified, the method used in the em- bodiment is a general technique in the field.

The endpoints and any values of the range disclosed herein are not limited to that exact range or value and shall be under- stood to contain values close to these ranges or values. For nu- merical ranges, between endpoint values of each range, between endpoint values and individual point values, and individual point values may be combined to obtain one or more new numerical ranges, which shall be regarded as specifically disclosed herein.

Raw materials are shown in Table 1.

Table 1 ee lactobacillus casei N16, N17, N40, build the key laboratory of functional dairy prod-

N31, N80, N88 ucts sacA-F/R Sangon Biotech {Shanghai) Co., Ltd.

ExTaqHSenzyme | Takara Biomedical Technology (Beijing) Co., Ltd.

Lactose and sucrose standards | Sigma Aldrich (USA)

Experimental instruments are shown in Table 2.

Table 2 ==

HPX-87P (300 mmx7.8 mmx9 ym)

Bio Rad (USA) chromatographic column

Waters (USA)

Index Detector (RID)

Embodiment

This embodiment provided a method for screening a lactobacil- lus casei fermentation agent metabolizing sucrose, comprising the following steps:

Sl, amplification of a sacA gene: extracting genomic DNA of the lactobacillus casei to be test- ed, and obtaining a full-length sequence of the sacA gene of the lactobacillus casei to be tested through amplification.

Specific operation steps of genomic DNA included: inoculating the lactobacillus casei in an MRS liquid culture medium according to 1% (v/v) inoculation, and allowing same to stand at 37+1°C for culture; when OD600 = 0.8-1.0, taking 2.5 mL of bacterial liquid, performing centrifugation for 5 min at 4°C and 12000xg, and remov- ing supernatant; adding 2 mL of a TES solution to resuspend the bacteria, performing centrifugation for 5 min at 4°C and 12000xg; adding 200 pL of 50mg/mL lysozyme to resuspend the bacteria, and taking 200 g of the mixture, and performing digestion for 1.5h at 37°C; adding 20 pL of RNase (10 mg/mL), gently performing well mix- ing, and placing the mixture for 5 min at room temperature; adding 20 pL of a protease K solution, and gently performing well mixing; adding 220 pL of a buffer solution GB, performing oscillation for 15s, performing treatment for 10 min in a 70°C water bath, and per- forming short centrifugation; adding 220 uL of anhydrous ethanol, and performing well mixing for 15s; performing centrifugation for 30s at 12000xg, and pouring waste liquid; adding 500 pL of a buff- er solution GD, performing centrifugation for 30s at 12000xg, and pouring waste liquid; adding 700 pL of a washing solution PW, performing centrifugation for 30s at 12000xg, and then adding 500 nL of the washing solution PW for washing; performing centrifuga- tion for 2 min at 12000xg, placing the mixture for 7 min at room temperature, and drying the residual washing solution in an ad- sorbing material; and dropwise adding 50 uL of a TE buffer solu- tion preheated at 65°C-70°C, performing centrifugation for 2 min at 12000xg, then collecting samples, and preserving the samples at - 20°C for later use.

A formula of the MRS liquid culture medium included: dissolv- ing 5 g of anhydrous sodium acetate, 2 g of ammonium citrate diba- sic, 2 g of potassium phosphate dibasic, 0.58 g of magnesium sul- fate, 0.19 g of manganese sulfate tetrahydrate, 1mL of tween-80, 20 g of glucose, 10 g of peptone, 5 g of yeast extract powder, and 5 g of beef extract powder in 900 mL of deionized water, regulat- ing a pH value to 6.5 with 1 mol/L of sodium hydroxide, and making up to 1 L; and performing autoclaved sterilization for 15 min at 121°C.

The TES solution was prepared by adding 25 g of sucrose, 5 mL of a Tris-Cl mother solution (1 M, pH=8.0), and 6 mL of an EDTA mother solution (0.5 M, pH=8.0), regulating pH to 8.0, making up to 100 mL with deionized water, performing autoclaved steriliza- tion for 15 min at 121°C, and performing preservation at room tem- perature.

A sacA gene was amplified, sacA-F/R was used as a PCR primer, and nucleotide sequences of sacA-F and sacA-R were shown as SEQ ID

NO.1 and SEQ ID NO.2. sacA-F!:5'- CAAGGTGGTAAGTTCTGGGGGTAACATTTCAAACAAGST -3'; sacA-R:5!- GCTTGCGACAAGCCTTACGTTGTGGCGACA -3!.

The sacA gene is amplified by using a PCR technology; an Ex

Tag HS enzyme is selected to establish a 25 pL system; and PCR re- action conditions comprise: 95°C, 3min; [95°C, 30 s; 60°C, 30s; and 72°C, 2 min] x 30 cycles; 72°C, 5 min; and 4°C. 82, determination of an amino acid sequence of sucrose-&6- phosphohydrolase of the lactobacillus casei to be tested: based on the full-length sequence of the sacA gene acquired in 31, determining the amino acid sequence of the sucrose-6- phosphohydrolase of the lactobacillus casei to be tested through software; and 33, comparison of the amino acid sequence of the sucrose-6- phosphohydrolase comparing the amino acid sequence, which is shown as SEQ ID

NO.3, of the sucrose-6-phosphohydrolase of the lactobacillus casei to be tested obtained in S2, and determining that the lactobacil- lus casei to be tested is able to metabolize sucrose. 54, verification of utilization of the lactobacillus casei to be tested on sucrose:

S41, activating the lactobacillus casei to be tested:

The lactobacillus casei to be tested was inoculated in the

MRS according to a 1% (v/v) addition amount, and cultured at 37+1°C for 12h for strain activation.

S42, determining the utilization of the lactobacillus casei to be tested on the sucrose:

D Detection of growth of strains

Strains were inoculated in an S-MRS culture medium according to a final concentration of 1x10’ CFU/mL, ODss and pH values were measured every 2h, the growth of the strains was determined, and this experiment was performed for three biological replicates. 2) Determination of sucrose content in fermented milk 93% (w/w) raw milk and 5% (w/w) sucrose were mixed and homog- enized through 20MPa, pasteurization was performed for 5 min at 95°C, the milk was cooled to 37 °C, and a fermentation base materi- al was obtained through preparation. The lactobacillus casei to be tested was inoculated in the fermentation base material according to an addition of 5x10° CFU/g, and was cultured for 24h at 37°C, and 3 biological replicates were performed.

Accurately weighing a sucrose standard to a 100mL volumetric flask, making up to 100 mL with ultrapure water, wherein mass con- centrations of the sucrose respectively are 3, 2.5, 2, 1.75, 1.50, 1.25, 1.0, 0.75, 0.50 and 0.25 mg/mL; plotting a standard curve by using the concentration of the standard as a horizontal coordinate and using a peak area as a longitudinal coordinate. 5.0 g of the fermented milk was weighed and placed to a 100mL beaker, and 25.0 g of deionized water was added and fully dissolved by using a mag-

netic stirrer; 2.5 mL of a Carrez reagent A and 2.5mL of a Carrez reagent B were added in sequence and were fully dissolved for 30 min, so as to obtain a mixture; centrifugation was performed on the mixture for 15 min at 5000xg; supernatant was diluted to 100 mL with distilled water, and was filtered through a 0.22 um filter membrane; and in combination with a Bio-Rad Aminex® HPX-87P (300 mmx7.8 mmx9 um) chromatographic column and a 2414-type differen- tial Refractive Index Detector (RID), a 5mM sulfuric acid aqueous solution was used as a mobile phase, with a flow rate being 550 pL/min, a column temperature was set to 60 °C, a sample size was 2

HL, and lactose and sucrose content in samples was detected by us- ing an external standard method, so as to calculate a utilization rate of sucrose.

The Carrez reagent A was prepared by weighing 10.60 g of po- tassium ferrocyanide, and dissolving same to 100 mL with deionized water. The Carrez reagent B was prepared by weighing 21.90 g of zinc acetate dihydrate, dissolving same with the deionized water, adding 3 mL of acetic acid for dissolving, and making the mixture up to 100 mL.

In this embodiment, six lactobacillus casei strains were se- lected, respectively being LC N16, N17, N31, N40, N80, and N88.

Metabolic effects of the six strains on the sucrose were respec- tively determined according to the above method.

Result analysis:

The growth of each lactobacillus casei strain was shown in

Fig. 1. When the sucrose was a single sugar source, the growth of the LC N16, the N17, and the N40 entered a logarithmic phase at 4 hours, and entered a stable phase at 12 hours; and when the su- crose was the single sugar source, OD600 values of the LC N31, the

N80, and the N88 were all less than 0.250 within 16 hours, indi- cating that these three strains did not utilize the sucrose.

Amino acid sequences of the sucrose-6-phosphohydrolase SacA of the LC N16, N17, and N40 strains were compared with amino acid sequences of the sucrose-€-phosphohydrolase SacA of the LC N31,

N80, and N88 strains. Results were shown in Fig. 2. 5 amino acids (Valine, threonine, asparagine, leucine, and valine) of p- fructosidase SacA in the LC N16, the N17, and the N40 were differ-

ent from that in an LC group (Isceucine, alanine, serine, phenyl- alanine, isoleucine). When mutating from threonine to alanine, the polarity and charge of the amino acid was changed.

The amino acid sequence of the sucrose-6-phosphchydrolase

SacA of the lactobacillus casei utilizing the sucrose was shown as

SEQ ID NO.3. Specifically:

HFKOGGKFMKEATWSTAARYQPYSSWAPDY IMKLKAQVAAS—

KWRTKTHVQPDTGLINDPCSLNEEFNNKWHLYYQQFPEFGPVHGLKSWA—

HAVSKDLENWRRVPGDLLPDNEYDSHGAYTGSALVTHGTLRLMYTGNARDDOWHRHSTQL-

GAVLGADGRLFKDPKPLVLTPPTGYTQEFRDPFLENYEGQTYVLIGGQRPDHTGAIL-

LYAKQTDKSWREVAPLSIPDEFCGYMVECPNITFINGKVVLVYCPQGLDODEFPEYENVYP-

NIALVADSFDPTTGNLTHORLONIDKGEDEYATRLANTDDDGTLAISWLGLPDTTYPTDDDG-

WAGVLSYVROLTLRDDHVCLYPHPAIKNLRETAVEDLPVIQQHDDEWTVTNLEGAFELALT-

LAAGOKTTIHLPDGDHDOLLIHLDSDSGQGMIQRENRN-

NGGSLRQFGFPAGKTVEIRLEIDVSVFELFIDOGYRVVSGRFFGNEAPTAA-

RVTPPSAASDVVSWNLKKDNGGL.

Determination results of the sucrose content in the fermented milk were shown in Fig. 3. Sucrose consumed by the LC N16, N17, and N40 strains respectively was 1.2 g, 1.43 g, and 1.35 g, and sucrose utilization rates respectively were 24%, 28.6%, and 27%; and sucrose consumed by the LC N31, N80, and N88 strains respec- tively was 0.028 g, 0.027 g, and 0.029 g, and sucrose utilization rates respectively were 0.56%, 0.54%, and 0.58%. It was verified that the lactobacillus casei metabolizing sucrose was directional- ly screened by using the amino acid sequence of the sucrose-6- phosphohydrolase.

In summary the technical problem to be urgently solved ex- pected to use the lactobacillus casei metabolizing sucrose direc- tionally screened based on the amino acid sequence of the sucrose- 6-phosphohydrolase as a fermentation agent to apply to fermenta- tion of milk products, so as to accelerate a fermentation rate, thereby improving the quality of the fermented milk.

Finally, it should be noted that the above is only used to explain the technical scheme of the invention rather than the lim- itation, although the invention is explained in detail according to the better arrangement scheme, the ordinary technicians in the field should understand that the technical scheme of the invention can be modified or replaced, without breaking away from the spirit and scope of the technical scheme of the invention.

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Claims

CONCLUSIONS

A method for screening a lactobacillus casei fermentant that metabolizes sucrose, comprising the following steps: S1, amplification of a sacA gene: extracting genomic DNA from the lactobacillus casei to be tested and obtaining a complete sequence of the sacA gene of the Jactobacillus casei to be tested by amplification, wherein PCR primers amplifying the sacA gene contain nucleotide sequences shown as SEQ ID NO.1 and SEQ ID NO.2; S2, determination of an amino acid sequence of sucrose-6-phosphohydrolase of the lactobacillus casei to be tested: based on the complete sequence of the sacA gene obtained in S1, determination of the amino acid sequence of the sucrose-6-phosphohydrolase of the lactobacillus to be tested casei using software; and S3, comparison of the amino acid sequence of the sucrose-6-phosphohydrolase comparison of the amino acid sequence, shown as SEQ ID NO.3, of the sucrose-6-phosphohydrolase of the lactobacillus casei under test, obtained in S2, and finding that the lactobacillus casei to be tested is capable of metabolizing sucrose.

Method according to claim 1, further comprising the following steps: S4, checking the use of the lactobacillus casei to be tested on sucrose: S41, activating the lactobacillus casei to be tested; and S42, determining the use of the lactobacillus casei to be tested on sucrose.

The method of claim 1, wherein extracting the genomic DNA of the lactobacillus casei to be tested in S1 specifically comprises the following steps: inoculating the lactobacillus casei to be tested into a culture medium,

culturing the same in a stationary manner, when OD600 = 0.8-1. 0, take bacterial fluid for centrifugation, remove the supernatant, add TES solution to the precipitate to resuspend the bacteria, centrifuge, remove the supernatant, then add lysozyme to the bacteria to resuspend the bacteria , perform digestion, then add RNase to well mix, then add a protease K solution to well mix, add a buffer solution GB for oscillation, perform treatment in a water bath, add anhydrous ethanol to well mix, centrifuge and pour off waste liquid; add a buffer solution GD, centrifuge and pour off the waste liquid; adding a washing solution PW to wash, centrifuging and drying the remaining washing solution in an adsorbent material; and add a pre-warmed TE buffer solution drop by drop, centrifuge, collect samples and store the samples at a low temperature for later use.

Method according to claim 1, wherein the sacA gene is amplified in S1 using a PCR technology; an Ex Tag HS enzyme is selected to make a 25 pL system; and the PCR reaction conditions are: 95°C, 3min; [95°C, 30 s; 60°C, 30s; and 72°C, 2 min] x 30 cycles; 72°C, 5 min; and 4°C.

The method of claim 2, wherein in 54, S41, activating the lactobacillus casei to be tested comprises the following specific steps: inoculating the lactobacillus casei to be tested into an MRS liquid culture medium, and culturing the lactobacillus to be tested - Jus casei at 37+1°C for 12 hours, and carrying out strain activation.

The method of claim 2, wherein in S4, S42 determining the utilization of the lactobacillus casei to be tested on the sucrose comprises the following specific steps: first determining whether the lactobacillus casei to be tested is capable of utilizing the sucrose; the lactobacillus casei inoculum to be tested

learn to culture the lactobacillus to be tested in a liquid MRS culture medium for 12 hours at 37+1°C and detect the sucrose metabolism by the strain; then use the strain that has been determined to be able to utilize the sucrose as a basis for fermented milk of a fermentation agent and calculate the sucrose utilization rate; preparation of a fermented milk: mix raw milk and sucrose, homogenize and pasteurize, and after cooling prepare a fermentation raw material; and inoculate the activated lactobacillus casei to be tested into the fermentation raw material with an addition of 5x10° CFU/g, and culture at 37°C to obtain the fermented milk; and detection of the sucrose content: accurately weigh a sucrose standard in a 100 ml volumetric flask, make up to 100 ml with ultrapure water, in which the mass concentrations of the sucrose are 3, 2.5, 2, 1.75, 1.50 respectively. , 1.25, 1.0, 0.75, 0.50 and 0.25 mg/mL; plotting a sucrose standard in a 100 ml volumetric flask. 25 mg/mL; plot a standard curve using the concentration of the standard as the horizontal coordinate and a peak area as the longitudinal coordinate; weigh the fermented milk in a beaker and add deionized water to dissolve it with a magnetic stirrer; successively add a Carrez reagent A and a Carrez reagent B and obtain a mixture after complete dissolution; centrifuge the mixture and take the supernatant; dilute the supernatant with distilled water and filter using a filter membrane; in combination with a chromatographic column and a differential refractive index detector (RID), using a sulfuric acid solution as a mobile phase and detecting the sucrose content in a fermented milk sample using an external standard method, so that the sucrose utilization rate can be calculated.