CN108048342B - Pressure-resistant probiotics and food and preparation method thereof - Google Patents
Pressure-resistant probiotics and food and preparation method thereof Download PDFInfo
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- 230000000529 probiotic effect Effects 0.000 claims abstract description 23
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- 238000012216 screening Methods 0.000 abstract description 13
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Abstract
The invention discloses a pressure-resistant probiotic, food and a preparation method thereof, wherein the pressure-resistant probiotic is preserved in the China general microbiological culture Collection center and comprises Lactobacillus plantarum (Lactobacillus plantarum) with the number of CGMCC No.14398 and Lactobacillus curvatus (Lactobacillus curvatus) with the number of CGMCC No.14397, and the preparation method comprises the following steps: the method comprises the steps of seed selection, inoculation, pressure resistance screening, expanded culture and strain preservation to obtain pressure-resistant probiotics, and adding the pressure-resistant probiotics into food to obtain pressure-resistant probiotic food, wherein the pressure-resistant probiotics have certain activity in the food, and fermented food which is nutritional and safe and contains active probiotics can be obtained.
Description
Technical Field
The invention relates to the field of microorganisms, in particular to pressure-resistant probiotics, food and a preparation method thereof.
Background
Probiotic (Probiotic) has important physiological and health-care functions of nutrition, immunity enhancement, cholesterol reduction, generation of metabolites with bacteriostatic activity and the like, and is popular in the market in recent years. And is classified As Generally Recognized As Safe (GRAS) by the U.S. FDA, etc.
At present, a lot of foods are rich in active probiotics, but in the production process of the traditional sterilization and preservation processes such as heat sterilization or radiation sterilization, on one hand, harmful bacteria and probiotics are killed indiscriminately, and on the other hand, the problems of serious quality deterioration, heavy cooked taste, poor brittleness, nutrient loss and the like exist in the foods.
The information disclosed in this background section is only for enhancement of understanding of the general background of the invention and should not be taken as an acknowledgement or any form of suggestion that this information forms the prior art already known to a person skilled in the art.
Disclosure of Invention
The invention aims to provide pressure-resistant probiotics, food and a preparation method thereof, so that the pressure-resistant probiotics have certain activity in the food, and fermented food which is nutritional and safe and contains active probiotics is obtained.
In order to achieve the purpose, the invention provides pressure-resistant probiotics which are preserved in the common microorganism center of China Committee for culture Collection of microorganisms and comprises Lactobacillus plantarum (Lactobacillus plantarum) with the number of CGMCC No.14398 and Lactobacillus curvatus (Lactobacillus curvatus) with the number of CGMCC No. 14397.
The invention also provides a preparation method of the pressure-resistant probiotics, which comprises the following steps:
(1) seed selection: selecting probiotic strains with remarkable functions corresponding to the pressure-resistant probiotics, wherein the probiotic strains optionally comprise lactobacillus, bifidobacterium, streptococcus, lactococcus, leuconostoc and yeast except the probiotic strains corresponding to the pressure-resistant probiotics;
(2) inoculation: inoculating the probiotic strains into a liquid culture medium under an aseptic operation condition for culture;
(3) screening the pressure resistance: performing pressure acclimation on the probiotic strains obtained by inoculation in the step (2) through ultrahigh pressure, and screening pressure-resistant probiotics, wherein the ultrahigh pressure is adopted within the pressure range of 50-600MPa, and the treatment time is 1-60 min;
(4) and (3) amplification culture: inoculating the pressure-resistant probiotics screened in the step (3) to a liquid culture medium under the aseptic operation condition for amplification culture;
(5) and (3) strain preservation: and (3) preserving the pressure-resistant probiotics obtained by expanding culture in the step (4) under the aseptic operation condition.
The probiotic strains adopted in the step (1) also comprise undiscovered probiotic bacteria.
In another embodiment of the above method for preparing pressure-resistant probiotics, the culture conditions in step (2) are as follows: culturing at 37 +/-2 ℃ for 1-2 days; optionally, the liquid medium is MRS broth; further optionally, the thallus concentration in the obtained bacteria liquid reaches 10 through the step (2)7～109CFU/mL, wherein the calculating method of the thallus concentration is a plate counting method.
In another embodiment of the preparation method of the pressure-resistant probiotics, the pressure range used in the ultrahigh pressure in step (3) is 250-600 MPa.
In another embodiment of the preparation method of the pressure-resistant probiotics, the screening process in the step (3) is as follows: pressure acclimation is carried out on the probiotic strains within the pressure and time range adopted by ultrahigh pressure until the following indexes are reached: when the survival rate of the selected probiotics under a certain pressure value reaches 0.001-0.01%, the survival number of the selected probiotics reaches 105～106Or the survival rate logarithm can be obviously improved by 1-3 times compared with the survival rate logarithm of the last treatment condition.
In another embodiment of the above method for preparing pressure-resistant probiotics, the screening process of step (3) further comprises: (A) when the survival rate of the probiotics can reach the index defined by the screening process in the step (3), continuously increasing the pressure or prolonging the treatment time to continuously screen; and (B) when the survival rate of the probiotics fails to reach the index defined by the screening process in the step (3), repeatedly domesticating under the same pressure until the survival rate of the probiotics reaches 0.001-0.01%.
In another embodiment of the preparation method of the pressure-resistant probiotics, the temperature used for screening is normal temperature, the treatment time of the step (A) is 1-60min, and the acclimatization frequency of the step (B) is 1-500 times.
In another embodiment of the preparation method of the pressure-resistant probiotics, the condition for performing the expanded culture in the step (4) is to culture the pressure-resistant probiotics at 37 ± 2 ℃ for 1-2 d, optionally, the liquid culture medium is MRS broth, further optionally, the concentration of the obtained bacteria reaches 10 through the step (4)7～109CFU/mL, wherein the calculating method of the thallus concentration is a plate counting method.
In another embodiment of the above method for preparing pressure-resistant probiotics, the strain preservation conditions in step (5) are as follows: mixing the pressure-resistant probiotics obtained by the enlarged culture in the step (4) with 50% of sterile glycerol according to the proportion of 1:1, and storing at the temperature of-80 ℃.
The invention also provides a preparation method of the pressure-resistant probiotic food, which comprises the steps of inoculating the pressure-resistant probiotic into the food by adopting a probiotic food preparation technology and a probiotic food sterilization technology, and sterilizing by adopting the ultrahigh pressure.
The preparation method of the pressure-resistant probiotic food adopts a probiotic food preparation technology and a probiotic food sterilization technology, so that the pressure-resistant probiotics survive, harmful microorganisms which are not pressure-resistant in the food are killed, and the harmful microorganisms are killed in a targeted manner, so that the food rich in active probiotics is obtained.
MRS broth related to the present invention was purchased from Oboxing Biotechnology, Inc., Beijing; the ultra-high pressure is achieved by an ultra-high pressure treatment apparatus available from Stansted, UK under the product model number FPG 7100: 9/2C.
Compared with the prior art, the invention has the following beneficial effects: the ultrahigh pressure technology adopted by the application mainly acts on hydrogen bonds, can ensure that covalent bonds are intact, is used in a sterilization process of food delivery, directionally kills harmful microorganisms in food, realizes that the pressure-resistant probiotics have certain activity in food, can furthest retain the quality and flavor of the food, and is subsequently applied to the processing process of the food and combined with the ultrahigh pressure processing technology of the food to obtain the fermented food which is nutritional, safe and contains active probiotics.
Preservation information
The Lactobacillus plantarum (Lactobacillus plantarum) and the Lactobacillus curvatus (Lactobacillus curvatus) provided by the invention are already preserved in the China general microbiological culture Collection center in 7-10.2017, with the preservation addresses: xilu No.1 Hospital No. 3, Beijing, Chaoyang, North. And E, postcode: 100101, and the preservation numbers are CGMCC No.14398 and CGMCC No.14397 in sequence.
Drawings
Fig. 1 is a flow chart of probiotic pressure resistance screening according to the invention.
FIG. 2 is a graph showing the acclimatization of pressure resistance of Lactobacillus plantarum strain and Lactobacillus curvatus strain under an ultra-high pressure of 400MPa according to the present invention; wherein lg (N/N)0) Representing the logarithm of the survival rate, N is the number of viable bacteria after ultrahigh pressure treatment, N0The initial viable count of the strain.
Detailed Description
The following detailed description of the present invention is provided in conjunction with the accompanying drawings, but it should be understood that the scope of the present invention is not limited to the specific embodiments.
Example 1:
(1) seed selection: and selecting a lactobacillus plantarum strain.
(2) Inoculation: under the aseptic operation condition, inoculating the lactobacillus plantarum strain into an MRS broth culture medium, and culturing for 1-2 d at 37 +/-2 ℃ to ensure that the concentration of the strain in the strain liquid reaches 107～109CFU/mL。
(3) Screening the pressure resistance of lactobacillus plantarum: carrying out ultrahigh pressure test on the lactobacillus plantarum bacterial liquid, putting a proper amount of the bacterial liquid into an aseptic sealed packaging container, and treating the lactobacillus plantarum bacterial liquid by using an ultrahigh pressure treatment device according to 300MPa for 1min (acclimatization 2 generation), 300MPa for 3min (acclimatization 2 generation), 300MPa for 5min (acclimatization 1 generation), 400MPa for 1min (acclimatization 3 generation) and 400MPa for 3min (acclimatization 22 generation) to gradually improve the ultrahigh pressure treatment condition, so that the pressure resistance of the lactobacillus plantarum is improved, the survival rate of the lactobacillus plantarum is improved by 130 times compared with that of the common lactobacillus plantarum under the 400MPa condition, and the lactobacillus plantarum capable of resisting the 400MPa ultrahigh pressure treatment is obtained.
(4) And (3) carrying out expanded culture on pressure-resistant lactobacillus plantarum: under the aseptic operation condition, the lactobacillus plantarum processed in the step (3) is inoculated into MRS broth for culture for 1-2 d under the condition of 37 +/-2 ℃ so that the thallus concentration reaches 107～109CFU/mL。
(5) And (3) strain preservation: under the aseptic operation condition, the pressure-resistant Lactobacillus plantarum (Lactobacillus plantarum) in the step (4) is mixed with 50% of sterile glycerol according to the ratio of 1:1, and is stored at the temperature of minus 80 ℃, wherein the Lactobacillus plantarum (Lactobacillus plantarum) is stored in the China general microbiological culture Collection center (CGMCC for short) in 7, 10 and 2017, and the storage number of the Lactobacillus plantarum is CGMCC No. 14398.
Example 2:
(1) seed selection: selecting a Lactobacillus curvatus strain.
(2) Inoculation: under the aseptic operation condition, the Lactobacillus curvatus strain is inoculated into an MRS broth culture medium and cultured for 1-2 d at the temperature of 37 +/-2 ℃ to ensure that the concentration of the strain in the strain liquid reaches 107～109CFU/mL。
(3) Screening the pressure resistance of lactobacillus curvatus: carrying out ultrahigh pressure test on the lactobacillus flexus liquid, putting a proper amount of the liquid into an aseptic sealed packaging container, and treating the lactobacillus flexus liquid by using an ultrahigh pressure treatment device according to 300MPa for 1min (acclimatization 1 generation), 300MPa for 3min (acclimatization 3 generation), 300MPa for 5min (acclimatization 3 generation) and 400MPa for 1min (acclimatization 23 generation), so that the effect of gradually improving the ultrahigh pressure treatment condition to treat the lactobacillus flexus liquid is achieved, the pressure resistance of the lactobacillus flexus is improved, the survival rate of the lactobacillus flexus liquid is improved by 1157 times compared with that of the ordinary lactobacillus flexus under the 400MPa condition, and the lactobacillus flexus capable of enduring the ultrahigh pressure treatment of 400 MPa.
(4) And (3) carrying out expanded culture on pressure-resistant bent lactobacillus: subjecting the Lactobacillus curvatus treated in step (3) to aseptic processingThe expanded culture is inoculated into MRS broth and cultured for 1-2 d at the temperature of 37 +/-2 ℃ to ensure that the thallus concentration reaches 107～109CFU/mL。
(5) And (3) strain preservation: under the aseptic operation condition, the pressure-resistant Lactobacillus curvatus (Lactobacillus curvatus) in the step (4) is mixed with 50 percent of aseptic glycerin according to the proportion of 1:1, and the mixture is stored at the temperature of minus 80 ℃, and the Lactobacillus curvatus (Lactobacillus curvatus) is stored in China general microbiological culture Collection center (CGMCC for short) in 7-10 th in 2017, with the storage number of CGMCC No. 14397.
The data for screening the pressure resistance of the probiotics used in examples 1-2 are shown in Table 1, and the data for acclimatization are shown in FIG. 2.
TABLE 1 screening of the anti-stress Properties of Lactobacillus plantarum and Lactobacillus curvatus strains
As can be seen from the table 1, the survival rates of the two probiotics are gradually reduced along with the increase of the pressure and the time, but under the continuous domestication condition, the survival rates of the two probiotics are gradually improved under the same condition, so that the pressure resistance of the probiotics is improved, and the probiotics can be better applied to products.
The foregoing descriptions of specific exemplary embodiments of the present invention have been presented for purposes of illustration and description. It is not intended to limit the invention to the precise form disclosed, and obviously many modifications and variations are possible in light of the above teaching. The exemplary embodiments were chosen and described in order to explain certain principles of the invention and its practical application to enable one skilled in the art to make and use various exemplary embodiments of the invention and various alternatives and modifications as are suited to the particular use contemplated. It is intended that the scope of the invention be defined by the claims and their equivalents.
Claims (2)
1. The pressure-resistant probiotics is characterized in that the probiotics areLactobacillus curvatus (A)Lactobacillus curvatus) The strain is preserved in the China general microbiological culture Collection center with the preservation number of CGMCC No. 14397.
2. A method for preparing a pressure-resistant probiotic-containing food, characterized in that the pressure-resistant probiotic of claim 1 is inoculated into a food and sterilized with an elevated pressure of 400 Mpa.
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