CN112501079A - Lactic acid bacteria for controlling patulin and application of lactic acid bacteria in fruit juice - Google Patents
Lactic acid bacteria for controlling patulin and application of lactic acid bacteria in fruit juice Download PDFInfo
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- CN112501079A CN112501079A CN202011504112.2A CN202011504112A CN112501079A CN 112501079 A CN112501079 A CN 112501079A CN 202011504112 A CN202011504112 A CN 202011504112A CN 112501079 A CN112501079 A CN 112501079A
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Their preparation
- A23L2/70—Clarifying or fining of non-alcoholic beverages; Removing unwanted matter
- A23L2/84—Clarifying or fining of non-alcoholic beverages; Removing unwanted matter using microorganisms or biological material, e.g. enzymes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/31—Leuconostoc
- A23V2400/321—Mesenteroides
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Tropical Medicine & Parasitology (AREA)
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Abstract
The invention discloses a lactic acid bacterium for controlling patulin and application thereof, belonging to the technical field of biology; the lactobacillus is leuconostoc mesenteroides LB7, and the number of the preserved strains is CCTCC NO: m2020039 for the control of patulin in fruit juices; leuconostoc mesenteroides LB7 was used at a concentration of 1X 106cells/mL; preliminary experiments show that after LB7 treatment, the content of patulin in the experimental group is only 41% of that in the control group; the patulin in the grape juice can be completely degraded by inoculating the grape juice (the concentration of the patulin is 1 mu g/mL) with the inoculation amount of 5 percent for 72 hours; meanwhile, the bacterium has good control effect on the patulin in the apple juice and the peach juice, the final concentration of the patulin is 1 mu g/mL, after the cultivation for 72 hours, the concentrations of the patulin in the apple juice and the peach juice are respectively 0.32 mu g/mL and 0.35 mu g/mL, which are obviously lower than those of a controlAnd (4) grouping.
Description
Technical Field
The invention belongs to a method for biologically controlling toxins, and particularly relates to application of a lactic acid bacterium Leuconostoc mesenteroides subsp.mesenteroides LB7 for controlling patulin in fruit juice.
Background
Patulin is a toxic fungal secondary metabolite produced by metabolism of fungi such as penicillium, aspergillus and rhizopus, and is a genetic toxic compound. Paecilomyces patulinThe molecular formula of the element is C7H6O4The chemical name is: 4-hydroxy-4-hydro-furan (3, 2-carbon) pyran-2 (6-hydro) one. Toxicology tests show that patulin can affect fertility, has toxicological effects such as teratogenicity, carcinogenicity and immunotoxicity, is a neurotoxin, has wide and strong toxic effects on human and animals, can cause symptoms such as nausea, vomiting, hematochezia, fright syncope and coma due to acute toxicity, and is one of mycotoxins which have the greatest harm to human.
The contamination of food, such as fruits and crops, with patulin is quite common in the production and processing processes, and patulin is widely present in infected and rotten apples, grapes, peaches, corns, grains and other products, which not only causes great pollution to the environment and huge economic loss, but also poses great threat to the health of animals and people. With the continuous emergence of evidence of the damage of the patulin, relevant organizations of various countries have made restrictions on the content of the patulin in the food. The maximum allowable amount of patulin in apple and hawthorn products, fruit juice, jam and fruit wine specified in China is 50 mug/kg. The allied committees set more detailed standards specifying a maximum limit of 50ppb for patulin in fruit juices, especially apple juice and alcoholic beverages containing apple juice, a maximum limit of 25ppb for patulin in solid apple products, and a maximum limit of 10ppb for children's apple juice and infant food. The United States Food and Drug Administration (USFDA) also defines that the maximum content of patulin in apple juice is 50 μ g/L. With respect to these maximum limit regulations, the expert committee of the World Health Organization (WHO) in the Food and Agriculture Organization (FAO) has established a provisional limit standard for patulin, i.e. a maximum daily intake of 0.4 μ g patulin per kg body weight.
The current methods for controlling the content of patulin in fruits and their products mainly include physical methods and chemical methods. Physical methods refer to the use of various physical means to reduce the amount of decay of pathogenic molds and the production of patulin or to reduce the amount of patulin that has been produced during storage of the fruit. Such as: the generation of patulin is inhibited by regulating and controlling the storage conditions (low-temperature storage, air-conditioned storage and the like) of fruits and products, but the required equipment is large and the cost is higher; manual sorting and cleaning of fruits are enhanced, but a large amount of manpower is consumed, and the effect is general; physically adsorbing patulin in fruit products, but reducing the color and the phenol content of the fruit juice and seriously affecting the quality of the fruit juice; microwave treatment and irradiation treatment, ultrasonic treatment and the like of fruits and products, but equipment requirements are high and patulin in the fruits and the products cannot be completely removed. The chemical method for controlling patulin in fruits and fruit products mainly comprises the following steps: one is the use of various bactericides, such as: thiabendazole (thiabendazole), prochloraz (prochloraz), captan (captan), imazalil (imazalil), and the like, to control decay of fruits caused by pathogenic fungi. However, the widespread use of chemical fungicides has caused significant environmental pollution, and the problem of chemical fungicide residues has also posed a serious threat to the health of humans and animals. And secondly, the existing patulin in fruits and products is degraded by adopting the oxidative degradation of ozone and the degradation effect of sulfhydryl substances on the patulin, but the degradation only stays in an experimental level, whether the damage to the environment and human bodies is unknown, and the use safety is not guaranteed.
Domestic and foreign researches show that many antagonistic microorganisms including antagonistic bacteria, antagonistic yeast and antagonistic mold can be applied to fruits and products thereof, and are used for preventing and treating diseases caused by pathogenic mold and controlling patulin generated by pathogenic bacteria, and the biological prevention and treatment technology of pathogenic bacteria and the biological detoxification technology of patulin show good application prospects. However, the biological detoxification technology is still in a development stage, related researches at home and abroad are also in exploration and starting periods, and the related researches are incomplete and do not deeply limit the application of the biological detoxification technology in patulin control.
Disclosure of Invention
In view of the deficiencies of the prior art, the present invention is directed to solving one of the problems set forth above; the invention provides an apple surface separated from an ecological orchard, which is screened and separated from the apple surface of an agricultural ecological orchard in Zhenjiang City, cultured in an MRS culture medium at 25 ℃ and subjected to morphological observation; analyzing the 5.8S rDNA-ITS zone sequence of the strain, and carrying out molecular biological identification; identified as Leuconostoc mesenteroides subsp.mesenteroides LB7 (Leuconostoc mesenteroides subsp.mesenteroides LB7), abbreviated as Leuconostoc mesenteroides LB7(l.mesenteroides LB 7); researches prove that the strain has strong degradation effect on the patulin, can effectively control the patulin in a culture medium and fruit juice, and has potential application value.
The leuconostoc mesenteroides LB7(L.mesenteroides LB7) used in the invention is determined to have high safety and no harm to human body through ICR mouse acute toxicity test.
The invention firstly provides a bacterial strain for degrading patulin, which is preserved in China Center for Type Culture Collection (CCTCC) in Wuhan, the preservation date is 2020, 1 and 13 days, and the preservation number is as follows: CCTCC NO: m2020039, the proposed classification was named Leuconostoc mesenteroides subsp.mesenteroides LB7 (Leuconostoc mesenteroides LB 7).
The invention also provides a purpose of degrading patulin in fruit juice by using leuconostoc mesenteroides LB7, which is carried out according to the following steps:
(1) firstly, culturing and activating leuconostoc mesenteroides LB7 in an MRS solid culture medium; then selecting a single colony of leuconostoc mesenteroides LB7 growing in an MRS solid culture medium, inoculating the single colony into an MRS liquid culture medium for culture, centrifuging the culture to obtain thalli, cleaning the thalli for a plurality of times by using sterile distilled water, and diluting the thalli by using the sterile distilled water after cleaning to prepare the thalli into 1 × 106cell/mL of bacterial suspension;
(2) and (2) inoculating the bacterial suspension prepared in the step (1) into the fruit juice, and performing shake culture to realize the purpose of controlling patulin in the fruit juice.
Preferably, the MRS solid medium in step (1) has the following components: calculated by 1000mL, 10.0g of beef extract, 5.0g of yeast extract, 10.0g of peptone, 20.0g of glucose and K2HPO42.0g, sodium acetate 5.0g, MgSO4·7H2O 0.2g，MnSO4·4H20.05g of O, 801.0 g of Tween, 2.0g of triammonium citrate, 15.0g of agar and distilled water till 1000 mL.
Preferably, as in step (1)The MRS liquid culture medium comprises the following components: calculated by 1000mL, 10.0g of beef extract, 5.0g of yeast extract, 10.0g of peptone, 20.0g of glucose and K2HPO42.0g, sodium acetate 5.0g, MgSO4·7H2O 0.2g，MnSO4·4H2O0.05 g, Tween 801.0 g, triammonium citrate 2.0g, and distilled water to 1000 mL.
Preferably, the temperature of the culture activation in the step (1) is 25-28 ℃, and the time is 48-72 hours;
preferably, the conditions for inoculating the single colony in the PM medium for culture in the step (1) are as follows: shaking table cultivation at 180rpm, wherein the temperature is 25-28 ℃ and the time is 48-72 h; the dosage of the inoculation is as follows: each 50mL of MRS liquid medium was inoculated with 2-3 ring single colonies.
Preferably, the inoculation amount of the bacterial suspension inoculated into the fruit juice in the step (2) is 5-6%; namely the fungus suspension is 5 to 6 percent of the volume of the fruit juice.
Preferably, the fruit juice in step (2) comprises apple juice, grape juice or peach juice.
The invention has the advantages that:
(1) the leuconostoc mesenteroides LB7 used in the invention can efficiently control the accumulation of patulin in the culture medium, and after 72 hours of mixed culture, the result shows that when the content of the patulin in a control group is 0.77 mu g/g, the content of the patulin in an experimental group is only 0.33 mu g/g.
(2) The leuconostoc mesenteroides LB7 used by the invention can efficiently control the content of patulin in various fruit juices including apple juice, grape juice and peach juice; in apple juice, after 72 hours of culture of leuconostoc mesenteroides LB7, the concentration of patulin in the experimental group is only 0.32 mu g/mL, which is 34% of that in the control group; in grape juice, leuconostoc mesenteroides LB7 can completely degrade patulin in 72 hours, and the degradation rate reaches 100%; in peach juice, by 72 hours, the concentration of patulin in the peach juice added with leuconostoc mesenteroides LB7 is only 42 percent of that of a control group, and the concentration of the patulin is reduced to 0.35 mu g/mL.
(3) The leuconostoc mesenteroides LB7 used in the invention is determined to have high safety and no harm to human body through ICR mouse acute toxicity test, so that the leuconostoc mesenteroides LB7 can be applied to control patulin pollution in food, feed and products thereof, particularly various fruit juices and the like, and guarantee the edible safety of the food, the products thereof and the like.
Drawings
FIG. 1 is a graph of the effect of Leuconostoc mesenteroides LB7 on patulin control in MRS liquid medium.
FIG. 2 is a graph of the effect of Leuconostoc mesenteroides LB7 on patulin control in apple juice.
FIG. 3 is a graph showing the effect of Leuconostoc mesenteroides LB7 on patulin control in grape juice.
FIG. 4 is a graph showing the effect of Leuconostoc mesenteroides LB7 on the degradation of patulin in peach juice.
Detailed Description
The invention will be explained in more detail by means of the following examples; the following examples are illustrative only, and the present invention is not limited by these examples.
The apple juice, the grape juice and the peach juice used by the invention are all purchased from Zhenjiang European supermarket, and the fruit juice brand is convergent fruit juice.
Example 1:
the effect of leuconostoc mesenteroides LB7 on controlling patulin in MRS liquid culture medium;
(1) firstly, culturing and activating leuconostoc mesenteroides LB7 in an MRS solid culture medium; then selecting a single colony of leuconostoc mesenteroides LB7 growing in an MRS solid culture medium, inoculating the single colony into an MRS liquid culture medium for culture, centrifuging the culture to obtain thalli, cleaning the thalli for a plurality of times by using sterile distilled water, and diluting the thalli by using the sterile water after cleaning to prepare the product with the volume of 1 multiplied by 106cell/mL of bacterial suspension;
(2) after the strain culture is finished, adding patulin into a 50mL conical flask added with 20mL of liquid culture medium of the LMRS, adjusting the final concentration to be 100ng/mL, and establishing an experimental group (LB7) and a control group (CK); experimental group (LB7) was supplemented with 1mL of 1X 106cells/mL of lactic acid bacteria; 1mL of sterile water was added to the control group; placing into a shaking table, and culturing at 25 deg.C and rotation speed of 180rpm for 72 h; seven replicates of each treatment and two replicates of the entire experiment。
(3) Sample extraction and purification: 20mL of samples are respectively taken after 0h (mixed, namely, sampling is carried out, and is recorded as 0h), 12h, 24h, 36h, 48h, 60h and 72h of culture, the samples are poured into a pear-shaped separating funnel, equal volume of ethyl acetate is added, shaking is carried out for 5min, and standing is carried out for 3 min. After layering, using a graduated pipette to transfer the upper organic phase into another separating funnel, adding ethyl acetate with the same volume in the pear-shaped separating funnel into the funnel, and repeating the shaking extraction process. Discarding the water layer, combining the two ethyl acetate extracting solutions in a separating funnel, adding 10mL of sodium carbonate aqueous solution, immediately shaking, standing for layering, and finishing the purification operation within 2 min; extracting sodium carbonate water layer with 10mL ethyl acetate once, discarding sodium carbonate water layer, mixing ethyl acetate extractive solutions, adding 5 drops of glacial acetic acid, transferring to rotary evaporator, evaporating at 40 deg.C, evaporating to dryness, dissolving residue with 1mL ethyl acetate, filtering with 0.22 μm filter membrane, and determining with high performance liquid chromatography.
And (4) determining the result: the control effect of leuconostoc mesenteroides LB7 on patulin in MRS liquid culture medium is shown in figure 1; as can be seen from FIG. 1, the content of patulin in the medium supplemented with Leuconostoc mesenteroides LB7 was significantly reduced after 72 hours. Leuconostoc mesenteroides LB7 initial concentration is 1X 106At cells/mL, the patulin concentration in the medium decreased slowly at 0-24h compared to the CK group. At 24-48h, the concentration of patulin in the group added with leuconostoc mesenteroides LB7 is obviously reduced to be only 59 percent of the original concentration. Thereafter, the concentration of patulin continued to decrease, and at 72 hours, the concentration of patulin in the group to which Leuconostoc mesenteroides LB7 was added was only 0.34. mu.g/mL.
Example 2:
the control effect of leuconostoc mesenteroides LB7 on patulin in apple juice;
the operation steps are the same as (1) to (3) in the example 1, except that the MRS liquid culture medium in the step (2) is replaced by apple juice;
and (4) determining the result: the effect of Leuconostoc mesenteroides LB7 on patulin control in apple juice is shown in FIG. 2; as can be seen from FIG. 2, the application of Leuconostoc mesenteroides LB7 in apple juice was used to study the effect of Leuconostoc mesenteroides LB7 in controlling patulin in apple juice. As can be seen from FIG. 2, Leuconostoc mesenteroides LB7 also has a good control effect on patulin in apple juice. Compared with the CK group, the concentration of patulin in the apple juice added with leuconostoc mesenteroides LB7 was gradually reduced from 0h and was significantly lower than that of the control group from 12 hours. By 72 hours, the concentration of patulin in the test group was only 0.32. mu.g/mL, which is 34% of that in the CK group. The addition of leuconostoc mesenteroides LB7 can well control the penicillins in the apple juice.
Example 3:
the control effect of leuconostoc mesenteroides LB7 on patulin in grape juice;
the operation steps are the same as (1) to (3) in example 1, except that the MRS liquid culture medium in step (2) is replaced by grape juice;
and (4) determining the result: the control effect of Leuconostoc mesenteroides LB7 on patulin in grape juice is shown in FIG. 3; as shown in FIG. 3, in grape juice, Leuconostoc mesenteroides LB7 could completely degrade patulin at 72 hours, while at the same time, the content of patulin in the control group was not significantly changed, and was 0.82. mu.g/mL. From 24 hours later, the concentration of patulin in the grape juice added with leuconostoc mesenteroides LB7 was significantly reduced, and to 60 hours, the concentration of patulin in the grape juice added with leuconostoc mesenteroides LB7 was only 61% of that of the CK group. By 72 hours, the patulin in the grape juice added with leuconostoc mesenteroides LB7 was lower than the lowest detection limit value, and the patulin in the grape juice was completely degraded.
Example 4:
degradation effect of leuconostoc mesenteroides LB7 on patulin in peach juice;
the operation steps are the same as those of (1) to (3) in example 1, except that the MRS liquid culture medium in the step (2) is replaced by peach juice;
and (4) determining the result: the control effect of Leuconostoc mesenteroides LB7 on patulin in peach juice is shown in FIG. 4; as shown in FIG. 4, in peach juice, the concentration of patulin can be significantly reduced by adding Leuconostoc mesenteroides LB 7. At 12 hours, the concentration of patulin in peach juice to which Leuconostoc mesenteroides LB7 was added was only 0.71. mu.g/mL, while the concentration of patulin in the CK group was 0.88. mu.g/mL. Thereafter, the concentration of patulin in the experimental group was lower than that in the control group. From 24 hours later, the concentration of patulin in the peach juice added with leuconostoc mesenteroides LB7 is obviously reduced compared with that of the control group, and by 72 hours, the concentration of the patulin in the peach juice added with leuconostoc mesenteroides LB7 is only 42 percent of that of the control group, and the concentration of the patulin is only 0.35 mug/mL.
Description of the drawings: the above embodiments are only used to illustrate the present invention and do not limit the technical solutions described in the present invention; thus, while the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted; rather, it is intended that all such modifications and variations be included within the scope of the following claims and their equivalents.
Claims (9)
1. A lactic acid bacterium for controlling patulin, which is characterized in that the lactic acid bacterium is Leuconostoc mesenteroides (Leuconostoc mesenteroides subsp. mesenteroides) LB7 with the deposit number: CCTCC NO: M2020039.
2. Use of Leuconostoc mesenteroides of claim 1 for the control of patulin in culture medium or fruit juice.
3. Use of leuconostoc mesenteroides according to claim 2, characterized in that the fruit juice comprises apple juice, grape juice or peach juice.
4. Use of leuconostoc mesenteroides according to claim 2 for the control of patulin in fruit juices, which is carried out according to the following steps:
(1) firstly, culturing and activating leuconostoc mesenteroides LB7 in an MRS solid culture medium; then selecting leuconostoc mesenteroides LB growing in MRS solid culture medium7 inoculating single colony into MRS liquid culture medium, culturing, centrifuging to obtain thallus, washing with sterile distilled water for several times, diluting with sterile water to obtain 1 × 10 thallus6cell/mL of bacterial suspension;
(2) and (2) inoculating the bacterial suspension prepared in the step (1) into the fruit juice, and performing shake culture to realize the purpose of controlling patulin in the fruit juice.
5. The use of Leuconostoc mesenteroides for controlling patulin in fruit juice as claimed in claim 4, wherein the MRS solid medium in step (1) has the following composition: calculated by 1000mL, 10.0g of beef extract, 5.0g of yeast extract, 10.0g of peptone, 20.0g of glucose and K2HPO42.0g, sodium acetate 5.0g, MgSO4·7H2O 0.2g，MnSO4·4H20.05g of O, 801.0 g of Tween, 2.0g of triammonium citrate, 15.0g of agar and distilled water till 1000 mL.
6. The use of Leuconostoc mesenteroides for controlling patulin in fruit juice as claimed in claim 4, wherein the MRS liquid medium in step (1) has the following composition: calculated by 1000mL, 10.0g of beef extract, 5.0g of yeast extract, 10.0g of peptone, 20.0g of glucose and K2HPO42.0g, sodium acetate 5.0g, MgSO4·7H2O 0.2g，MnSO4·4H2O0.05 g, Tween 801.0 g, triammonium citrate 2.0g, and distilled water to 1000 mL.
7. The use of Leuconostoc mesenteroides of claim 4 for controlling patulin in fruit juice, wherein the temperature for culturing and activating in step (1) is 25-28 ℃ and the time is 48-72 h.
8. The use of Leuconostoc mesenteroides as claimed in claim 4 for controlling patulin in fruit juice, wherein the conditions for inoculating the single colony into PM medium for culturing in step (1) are as follows: shaking table cultivation at 180rpm, wherein the temperature is 25-28 ℃ and the time is 48-72 h; the dosage of the inoculation is as follows: each 50mL of MRS liquid medium was inoculated with 2-3 ring single colonies.
9. The use of Leuconostoc mesenteroides as claimed in claim 4 for controlling patulin in fruit juice, wherein the inoculation amount of the bacterial suspension inoculated into the fruit juice in the step (2) is 5-6%; namely the bacterial suspension is 5 to 6 percent of the volume of the fruit juice.
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Non-Patent Citations (1)
| Title |
|---|
| GUILLAUME LEGRAND NGOLONG NGEA 等: "Leuconostoc mesenteroides subsp. mesenteroides LB7 isolated from apple surface inhibits P. expansum in vitro and reduces patulin in fruit juices", 《INTERNATIONAL JOURNAL OF FOOD MICROBIOLOGY》 * |
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| CN113558159A (en) * | 2021-07-09 | 2021-10-29 | 西北农林科技大学 | Preparation process of fermented apple juice |
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