CN114190433B - Method for inhibiting acidification after yoghurt - Google Patents
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Abstract
The invention discloses a method for inhibiting acidification after yoghurt. The method comprises up-regulating the expression of acidifying gene LDB_RS05285 of Lactobacillus bulgaricus ATCC 11842 after fermentation of yogurt, up-regulating by adding Zn 2+ In the form of (a). The invention utilizes LDB_RS05285 gene to screen the food additive with high expression, and carries out the compounding of the food additive. The food additive not only has the effect of inhibiting acidification after yoghurt, but also can improve the stability of yoghurt. The compound food additive developed by the invention can effectively reduce the post-acidification problem in the storage process of the yoghurt, improve the storage stability of the yoghurt, prolong the shelf life of the yoghurt and provide a certain thought for the development of the food additive for inhibiting the post-acidification.
Description
Technical Field
The invention belongs to the technical field of food science, and particularly relates to a method for inhibiting acidification after yoghurt by regulating a gene LDB_RS05285.
Background
The yoghurt is a dairy product which takes fresh milk as a raw material, is added with a ferment and other ingredients, and is cooled and filled after fermentation. After the milk is fermented by lactic acid bacteria, the milk has the unprecedented effects, such as relieving lactose intolerance. The probiotics in the yoghurt also plays an important health care role in improving the gastrointestinal tract function of human bodies, resisting tumors, delaying aging, promoting human body absorption and the like. Research shows that the pH value of the fermented milk continuously decreases in the storage process, and the phenomena of too heavy sour taste, whey precipitation, sensory quality reduction and the like occur, which are caused by acidification after the occurrence of the fermented milk. Post-acidification of fermented milk refers to the phenomenon that bacteria are still growing and propagating during the process of storage, transportation, sales and eating of yoghurt after fermentation is finished, so that the fermented milk cannot be accepted by consumers due to too high acidity and reduced organoleptic quality, and the phenomenon is called post-acidification of yoghurt.
Lactobacillus delbrueckii subsp bulgaricus (Lactobacillus delbrueckii subsp. Bulgaricum) is also called lactobacillus bulgaricus, is widely applied to the fermented dairy industry due to excellent fermentation performance, can ferment macromolecular nutrient substances into micromolecular nutrient substances and endow products with special flavor, for example, can ferment lactose to generate glucose and galactose which are easy to be absorbed by human bodies, and can relieve lactose intolerance to a certain extent. In addition, l.bulgaricus also has effects of regulating gastrointestinal tract, inhibiting growth of harmful bacteria, enhancing immunity, etc. However, l.bulgarisus has a key problem in the fermentation industry that restricts the development of the industry, namely post acidification. In recent years, research shows that in the fermentation of yoghourt, the control of the growth of the lactobacillus bulgaricus is a key for solving post-acidification, and the students control the growth and the acidogenesis of the lactobacillus bulgaricus through methods of pasteurization, strain mutagenesis, supplementation of antibacterial substances and the like. Both of these methods can alleviate the progress of acidification after yogurt, however the effect is not absolutely inhibited.
Disclosure of Invention
The invention aims to provide a method for inhibiting acidification after yoghurt by regulating a gene LDB_RS05285.
The invention discloses a post-acidification key gene LDB_RS05285 of lactobacillus bulgaricus, which is obtained by adopting the following method:
(1) Inoculating Lactobacillus bulgaricus ATCC 11842 and placing the Lactobacillus bulgaricus ATCC 11842 and MRS culture medium in a 42 ℃ incubator, culturing to the end of logarithmic phase, and extracting bacterial genome;
(2) Designing an LDB_RS05285 specific primer to amplify a target gene, adding a tail to react, connecting a pMD19-T vector, screening positive clones, extracting plasmids, performing enzyme digestion verification and sequencing;
(3) Detecting the expression quantity of a target gene in the acidification process after yoghurt by using an RT-qPCR technology;
(4) Constructing a gene over-expression vector, introducing the recombinant vector into host bacteria by an electrotransformation method, and screening positive clones;
(5) Detecting the expression quantity of a target gene in recombinant bacteria by using an RT-qPCR technology;
(6) The application of the gene in the acidification process after yoghurt is verified.
A method for inhibiting acidification after yoghurt, which is to up-regulate the expression of an acidification gene LDB_RS05285 after lactobacillus bulgaricus ATCC 11842 after the fermentation of yoghurt is finished.
The expression method of the up-regulating gene LDB_RS05285 is to add Zn 2+ Or Ninsin.
Zn 2+ The optimal adding time is 7.5mg/kg of optimal adding concentration after fermentation.
Zn 2+ The most suitable adding mode is zinc gluconate.
A food composition additive comprises whey protein powder, skim milk powder and zinc gluconate (250-300): (200-250):1. The weight percentage of the food additive in the reconstituted skim milk is 2% -3%.
The application of the food additive in the process of delaying acidification after yoghurt.
The invention has the beneficial effects that: according to the invention, the LDB_RS05285 gene overexpression vector is constructed, and the recombinant vector is introduced into host bacteria by an electrotransformation method, so that the LDB_RS05285 gene overexpression is verified to be capable of effectively inhibiting acidification after yoghurt. The invention utilizes LDB_RS05285 gene to screen the food additive with high expression, and carries out the compounding of the food additive. The food additive not only has the effect of inhibiting acidification after yoghurt, but also can improve the stability of yoghurt. The compound food additive developed by the invention can effectively reduce the post-acidification problem in the storage process of the yoghurt, improve the storage stability of the yoghurt, prolong the shelf life of the yoghurt and provide a certain thought for the development of the food additive for inhibiting the post-acidification.
Drawings
FIG. 1 shows the measurement of the expression level of a target gene in a recombinant strain.
Figure 2 is the effect of recombinant strains on the post-yoghurt acidification process.
FIG. 3 shows the effect of different additives on the relative expression level of the gene LDB-RS 05285 in Lactobacillus bulgaricus ATCC 11842.
FIG. 4 shows the effect of zinc ion treatment at different concentrations on the relative expression level of gene LDB-RS 05285 in Lactobacillus bulgaricus ATCC 11842.
FIG. 5 is Zn 2+ Effect of different addition times on pH and acidity at 14d during storage of yoghurt.
FIG. 6 is Zn 2+ Effect of different addition concentrations on pH and acidity at 14d during storage of yoghurt.
FIG. 7 is Zn 2+ Effect of different addition modes on pH and acidity of 14d during storage of yoghurt.
FIG. 8 shows addition of Zn at room temperature of 20 ℃ 2+ Change in acidity value during storage compared to control.
FIG. 9 shows whey protein powder, skim milk and Zn 2+ The effect on pH and acidity at 14d during yogurt storage was compounded.
FIG. 10 shows the addition of Zn to a commercially available yogurt 2+ Change in acidity value during storage compared to control.
Detailed Description
The present invention will be described more fully hereinafter in order to facilitate an understanding of the present invention. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
Example 1 construction of LDB_RS05285 Gene overexpression vector
Based on the published gene sequence of LDB_RS05285 in the genome of Lactobacillus bulgaricus (Lactobacillus delbrueckii subsp. Bulgaricus) ATCC 11842 on NCBI, the DNAMAN 6.0 software was used to design the primers for the upstream and downstream of the target gene:
LDB_RS05285-F：5-GACGAGCTCGCATGGAAAATAAAGAGGACC-3；
LDB_RS05285-R：5-AAAACTGCAGCATTGGATTACACGTTTTTAAT-3。
amplifying a target gene by using a PCR technology, adding an 'A' reaction by using common Taq enzyme after the amplification is finished, purifying after the reaction is finished, connecting a cloning vector pMD19-T, constructing a pMD19-LDB_RS05285 plasmid, transforming escherichia coli DH5a competent cells, screening positive cloning strains, and sequencing. Extracting plasmids pMD19-LDB_RS05285 and pMG36e, simultaneously carrying out double digestion on the two plasmids by using PstI and SacI, recovering digestion products, connecting by using T4 DNA Ligase, transferring into competent cells of escherichia coli DH5a again, screening positive clones, and carrying out sequencing comparison. After the sequencing result is correct, extracting plasmid pMG36e-LDB_RS05285 by shaking, transferring the recombinant plasmid into competent cells of lactococcus lactis ML23 by using an electrotransfer instrument, culturing at 30 ℃, selecting the transformant for PCR verification, and detecting that the fragment size is correct by electrophoresis, namely the recombinant bacterium ML23-36e-LDB_RS05285. And (3) measuring the expression quantity by adopting qRT-PCR technology.
As a result, the target gene as shown in FIG. 1 was expressed in both the recombinant strain (ML 23-36 e-LDB-RS 05285) and the control strain (ML 23-36 e) and the expression level of the recombinant strain was significantly higher than that of the control strain (P < 0.05)
EXAMPLE 2 Effect of recombinant strains on post-yoghurt acidification Process
Inoculating activated Lactobacillus bulgaricus ATCC 11842 into skimmed milk culture medium, culturing at 37deg.C for 11 hr until skimmed milk solidifies, pasteurizing, inactivating lactobacillus in yogurt, cooling to room temperature, inoculating the pre-cultured LDB_RS05285 gene overexpression strain and control strain into inactivated yogurt respectively with the same bacterial amount, and refrigerating at 4deg.C for 0d, 1d, 2d, 3d, 4d, 5d, 6d, and 7d. The samples were taken on time to determine the number of viable bacteria and the pH.
As shown in FIG. 2, the number of viable bacteria gradually decreased with the storage time, the decrease of 0-3d was remarkable, and the decrease of 4-7d was gentle. In the late stage of storage, the total colony count of the strain over-expressed by the target gene is 92.3% (P > 0.05) of the control group. The pH of the control strain during the shelf-life of the yoghurt is significantly lower than that of the target gene over-expression strain (P < 0.05). It can be stated that the over-expression of the LDB_RS05285 gene reduces the post-acidification problem in the yoghurt storage process to a certain extent, improves the stability of the yoghurt storage and prolongs the shelf life of the yoghurt.
EXAMPLE 3 Effect of different addition measures on the expression level of the LDB-RS 05285 Gene in Lactobacillus bulgaricus ATCC 11842
Lactobacillus bulgaricus ATCC 11842 is inoculated into a liquid MRS culture medium conical flask with an initial pH value of 6.8 and 100mL at 1% (v/v), standing and culturing is carried out at 42 ℃ for 10h to mid-log phase, the temperature is 4 ℃, the speed is 6000r/min, centrifugation is carried out for 10min, 10mL of thalli are collected, the thalli precipitate is respectively placed into MRS culture media with different preselected food additives (with the same mass concentration) and the pH value of 4.8 for 40min, 1mL of thalli are collected by centrifugation, liquid nitrogen is frozen, and refrigeration is carried out at-80 ℃ for standby. Extracting total RNA of each frozen sample according to the specification method of the total RNA extraction kit of the United states Thermo Fisher Scientific TaKaRa RNAiso Plus; detecting the quality and the integrity of RNA of each sample by using 2% agarose gel electrophoresis; refer to PrimeScript TM RT reagent Kit with gDNA Eraser (Perfect Real Time) kit instruction method, removing genomic DNA from total RNA of each sample, reaction system: 5X gDNA Eraser Buffer. Mu.L, gDNA Eraser 1. Mu.L, total RNA 1. Mu.L, RNase Free dH 2 O7 μl; and synthesizing cDNA first strand of each sample, and reacting system: 10. Mu.L of the reaction solution, primeScript RT Enzyme Mix I. Mu.L, RT Primer Mix 1. Mu.L, 5X PrimeScript Buffer. Mu.L, RNase Free dH 2 O4. Mu.L. Freezing and storing the reverse transcription product at-20deg.C for use. The gene LDB_RS01640 is used as an internal reference gene, and the gene LDB_RS05285 is used as an internal reference gene, and qRT-PCR amplification is performed. qRT-PCR 20. Mu.L reaction system: 2X SYBR Green qPCR Master Mix. Mu.L, 1. Mu.L each of the upstream and downstream primers, 2. Mu.L of cDNA template, ddH 2 O6. Mu.L. qRT-PCR reaction procedure: activating Taq DNA polymerase activity at 95deg.C for 5 min; denaturation at 95℃for 5s, annealing at 51℃for 5s, extension at 72℃for 25s, 45 cycles total. Each sample reaction was repeated 3 times. According to 2 -ΔΔCT The relative expression of target gene is calculated by the method.
The experimental results are shown in FIG. 3, zn 2+ And Ninsin can obviously improve the expression of LDB_RS05285 gene, and in view of Ninsin as polypeptide, the Ninsin is not easy to store as commodity, and Zn is preferable 2+ As an additive to increase the expression of the LDB_RS05285 gene.
EXAMPLE 4 Effect of Zinc ion treatments at different concentrations on the relative expression level of the Gene LDB-RS 05285 in Lactobacillus bulgaricus ATCC 11842
Inoculating Lactobacillus bulgaricus ATCC 11842 into a conical flask with initial pH of 6.8 and 100mL liquid MRS culture medium at 1% (v/v), standing at 42deg.C for 10 hr to mid-log phase, centrifuging at 4deg.C for 10min at 6000r/min, collecting 10mL thallus, precipitating thallus, and placing different Zn respectively 2+ Treating in MRS culture medium with mass concentration (5 mg/kg,6.25mg/kg,7.5mg/kg,8.75mg/kg,10 mg/kg) and pH value of 4.8 for 40min, centrifuging to collect 1mL of thallus, freezing with liquid nitrogen, and refrigerating at-80deg.C for use. Extracting total RNA of each frozen sample according to the specification method of the total RNA extraction kit of the United states Thermo Fisher Scientific TaKaRa RNAiso Plus; detecting the quality and the integrity of RNA of each sample by using 2% agarose gel electrophoresis; refer to PrimeScript TM RT reagent Kit with gDNA Eraser (Perfect Real Time) kit instruction method, removing genomic DNA from total RNA of each sample, reaction system: 5X gDNA Eraser Buffer. Mu.L, gDNA Eraser 1. Mu.L, total RNA 1. Mu.L, RNase Free dH 2 O7 μl; and synthesizing cDNA first strand of each sample, and reacting system: 10. Mu.L of the reaction solution, primeScript RT Enzyme Mix I. Mu.L, RT Primer Mix 1. Mu.L, 5X PrimeScript Buffer. Mu.L, RNase Free dH 2 O4. Mu.L. Freezing and storing the reverse transcription product at-20deg.C for use. The gene LDB_RS01640 is used as an internal reference gene, and the gene LDB_RS05285 is used as an internal reference gene, and qRT-PCR amplification is performed. qRT-PCR 20. Mu.L reaction system: 2X SYBR Green qPCR Master Mix. Mu.L, 1. Mu.L each of the upstream and downstream primers, 2. Mu.L each of cDNAtemplate, ddH 2 O6. Mu.L. qRT-PCR reaction procedure: activating Taq DNA polymerase activity at 95deg.C for 5 min; denaturation at 95℃for 5s, annealing at 51℃for 5s, extension at 72℃for 25s for 45 cycles. Reactions were repeated 3 times per sample. According to 2 -ΔΔCT The relative expression of target gene is calculated by the method.
The experimental results are shown in FIG. 4, zn 2+ The expression level of the LDB_RS05285 gene was highest at a mass concentration of 7.5mg/kg.
EXAMPLE 5Zn 2+ Effect of different addition times on pH 14d and acidity during yogurt storage
Inoculating activated Lactobacillus bulgaricus ATCC 11842 into skimmed milk culture medium at 1% inoculum size, fermenting at 42deg.C for 11 hr to obtain curd, and adding Zn during fermentation for 0 hr, 4 hr, 8 hr, and 11 hr 2+ Then refrigerating at 4deg.C for 14d, measuring pH and acidity of the 14d during yogurt refrigerating, and examining Zn 2+ The effect of different addition times on inhibiting acidification after yoghurt.
The experimental results are shown in FIG. 5, zn 2+ The longer the fermentation time, the higher the pH, the lower the acidity and the greater the effect on inhibiting post-yoghurt acidification.
EXAMPLE 6Zn 2+ Effect of different addition concentrations on pH 14d and acidity during yogurt storage
Inoculating activated Lactobacillus bulgaricus ATCC 11842 into skimmed milk culture medium at 1% inoculum size, fermenting at 42deg.C for 11 hr, stopping fermentation, homogenizing cooled fermented milk, and adding Zn with different mass concentrations 2+ Refrigerating at 4deg.C for 14d, measuring pH and acidity of the 14d during yogurt refrigerating, and examining Zn with different mass concentrations 2+ The effect on inhibiting post-yoghurt acidification.
The experimental results are shown in FIG. 6, zn 2+ The higher the pH at a mass concentration of 7.5mg/kg, the lower the acidity, the greater the effect on inhibiting acidification after yoghurt, which is comparable to Zn 2+ The quality concentration is positively correlated with the up-regulation effect of the expression level of the LDB_RS05285 gene.
EXAMPLE 7Zn 2+ Influence of different addition modes on pH 14d and acidity during storage of yoghurt
Inoculating activated Lactobacillus bulgaricus ATCC 11842 into skimmed milk culture medium at 1% inoculum size, fermenting at 42deg.C for 11 hr, stopping fermentation, homogenizing cooled fermented milk, and adding Zn in different modes 2+ (grape)Zinc gluconate, zinc oxide, zinc lactate, and zinc sulfide), refrigerating at 4deg.C for 14d, measuring pH and acidity of the 14d during yogurt refrigerating, and examining Zn 2+ The effect of different addition modes of the acid milk on inhibiting acidification after the acid milk.
The experimental results are shown in fig. 7, and the added zinc gluconate has the highest pH, the lowest acidity and the greatest effect on inhibiting acidification after yoghurt.
Example 8 addition of Zn at ambient temperature of 20 ℃ 2+ Change in acidity value during storage as compared to control group
Inoculating activated Lactobacillus bulgaricus ATCC 11842 into skimmed milk culture medium at 1% inoculum size, fermenting at 42deg.C for 11 hr, stopping fermentation, homogenizing cooled fermented milk, and adding Zn with certain mass concentration 2+ (zinc gluconate), storing at 20deg.C, measuring acidity every 2 days, comparing with control group, and examining Zn 2+ The effect on inhibiting post-yoghurt acidification.
The experimental results are shown in FIG. 8, zn compared with the control 2+ The pH value of the post-acidification yoghurt can be obviously improved, and the acidity is reduced.
Example 9 addition of Zn to commercially available yogurt 2+ Change in acidity value during storage as compared to control group
The application experiments are carried out on three yogurt containing live lactobacillus bulgaricus bacteria on the market. The flavor fermented milk of the monarch Le Baoyi raw bacteria (shelf life of 21 days), the flavor fermented milk of the newly desired yoghurt workshop (shelf life of 21 days) and the flavor fermented milk of the illicit reconstituted milk (shelf life of 25 days). Selecting commercial yoghurt with fresh shelf life, and adding Zn with a certain mass concentration 2+ (zinc gluconate) and the acidity measured at different times.
The experimental results are shown in fig. 9, in which the acidity reached 120 ° at day 24 in the monarch Le Baoyi probiotic fermented milk blank group and 120 ° at day 30 in the treatment group (fig. 9A). The new hope was that the yogurt mill flavored fermented milk blank reached 120°t on day 30 and the treatment reached 120°t at 36 acidity (fig. 9B). Illicit reconstituted milk flavored fermented milk reached 120°t on day 30 and treatment group reached 120°t on day 32 (fig. 9C).
EXAMPLE 10 whey protein powder,Skim milk and Zn 2+ Compounding effects on pH 14d and acidity during yogurt storage
A food combination additive, wherein the mass ratio of whey protein powder, skim milk powder and zinc gluconate is 280:220:1. the weight percentage of the food additive in the reconstituted skim milk is 2.5%.
Inoculating activated Lactobacillus bulgaricus ATCC 11842 into fermented food composition culture medium (skimmed milk as control) at 1% inoculum size, fermenting at 42deg.C for 11 hr, stopping fermentation, homogenizing cooled fermented milk, refrigerating at 4deg.C for 14d, and measuring pH and acidity of the 14d during yogurt refrigerating.
As shown in fig. 10, the addition of the fermented food composition significantly increased the pH of the post-acidified yoghurt and decreased the acidity compared to the control group.
The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.
Claims (2)
1. A method for inhibiting acidification after yoghourt obtained by fermenting Lactobacillus bulgaricus ATCC 11842 is characterized in that zinc gluconate is added after the fermentation of yoghourt is finished to up-regulate the acidification gene after lactobacillus bulgaricus ATCC 11842LDB_RS05285The zinc gluconate added concentration is 7.5mg/kg.
2. The application of a food additive in delaying the post-acidification process of yoghurt obtained by fermenting lactobacillus bulgaricus ATCC 11842 is characterized in that the food additive is whey protein powder, skim milk powder and zinc gluconate, and the mass ratio of the whey protein powder to the skim milk powder to the zinc gluconate is (250-300): (200-250) 1; the weight percentage of the food additive in the reconstituted skim milk is 2% -3%.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102524421A (en) * | 2011-12-31 | 2012-07-04 | 黑龙江省完达山乳业股份有限公司 | Novel acid-base balance infant formula milk powder and preparation method thereof |
| WO2014192905A1 (en) * | 2013-05-31 | 2014-12-04 | 株式会社明治 | Fermented milk that does not undergo increase in acid level, and method for producing same |
| WO2017037052A1 (en) * | 2015-08-31 | 2017-03-09 | Chr. Hansen A/S | Lactobacillus fermentum bacteria inhibiting post-acidification |
| CN110117315A (en) * | 2019-06-12 | 2019-08-13 | 河北农业大学 | Post-acidification related gene of lactobacillus bulgaricus and application of post-acidification related gene in yoghourt |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102524421A (en) * | 2011-12-31 | 2012-07-04 | 黑龙江省完达山乳业股份有限公司 | Novel acid-base balance infant formula milk powder and preparation method thereof |
| WO2014192905A1 (en) * | 2013-05-31 | 2014-12-04 | 株式会社明治 | Fermented milk that does not undergo increase in acid level, and method for producing same |
| WO2017037052A1 (en) * | 2015-08-31 | 2017-03-09 | Chr. Hansen A/S | Lactobacillus fermentum bacteria inhibiting post-acidification |
| CN110117315A (en) * | 2019-06-12 | 2019-08-13 | 河北农业大学 | Post-acidification related gene of lactobacillus bulgaricus and application of post-acidification related gene in yoghourt |
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