CN110106112B - A kind of mixed probiotics of fermented vegetables, fermented vegetables and preparation method thereof - Google Patents
A kind of mixed probiotics of fermented vegetables, fermented vegetables and preparation method thereof Download PDFInfo
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Abstract
本发明涉及一种发酵蔬菜的混合益生菌、发酵蔬菜及其制备方法，属于蔬菜发酵技术领域。本发明所述发酵蔬菜的混合益生菌由Lactobacillus diolivorans V73和植物乳杆菌Lactobacillus plantarum V02组成。利用本发明所述混合益生菌发酵得到的发酵蔬菜具有调节肠道菌群的功能，预防心血管疾病，有利于人体健康，并且人工接种发酵蔬菜可强化发酵，降低亚硝酸盐含量，缩短发酵周期，提高产品质量，降低生产成本。The invention relates to a mixed probiotic for fermented vegetables, a fermented vegetable and a preparation method thereof, and belongs to the technical field of vegetable fermentation. The mixed probiotic bacteria of the fermented vegetables of the present invention is composed of Lactobacillus diolivorans V73 and Lactobacillus plantarum V02. The fermented vegetables obtained by fermenting the mixed probiotics of the present invention have the function of regulating intestinal flora, preventing cardiovascular diseases, and being beneficial to human health, and the artificial inoculation of fermented vegetables can strengthen fermentation, reduce the content of nitrite, and shorten the fermentation period , improve product quality and reduce production costs.
Description
Technical Field
The invention relates to the technical field of vegetable fermentation, in particular to mixed probiotics for fermented vegetables, the fermented vegetables and a preparation method thereof.
Background
The fermented vegetable is a traditional fermented food in China, has a long history, and is a vegetable product which is prepared by fermenting fresh vegetables in a closed jar through the change of microorganisms and enzymes in a complex microorganism system (carried in by raw materials) so as to greatly change the chemical components of a final product and the like. The fermented vegetable can prolong the storage period of the lettuce, is beneficial to transportation, is in balanced supply all the year round, improves the added value of the lettuce, can improve the flavor of the vegetable, and meets the increasing requirements of people on vegetable food. The existing vegetable fermentation method comprises natural fermentation and mother water fermentation, most of the traditional fermented foods are produced according to experience, so that the fermentation process is difficult to control, and the quality of the fermented vegetables is influenced by various factors such as the type of the fermented raw material vegetables, the salt concentration, the production season and the like. The fermented vegetables produced by the traditional fermentation method have the defects of long fermentation period, unstable quality, high salt content and the like.
Disclosure of Invention
The invention aims to provide mixed probiotics for fermented vegetables, the fermented vegetables and a preparation method thereof. The pickle pickled by the mixed probiotics has the functions of regulating intestinal flora, preventing cardiovascular diseases and being beneficial to human health, and the artificially inoculated fermented vegetables can strengthen fermentation, reduce the content of nitrite, shorten the fermentation period, improve the product quality and reduce the production cost.
The invention provides mixed probiotics for fermenting vegetables, which consist of Lactobacillus diolvorans V73 and Lactobacillus plantarum V02;
the biological preservation number of the Lactobacillus diolvorans V73 is CGMCC No. 16728;
the biological preservation number of the Lactobacillus plantarum V02 is CGMCC No. 16727.
The invention also provides a vegetable fermentation method based on the mixed probiotics in the technical scheme, which comprises the following steps:
inoculating the mixed probiotics in the technical scheme into a mixed solution of pepper water and salt to obtain a fermentation solution, mixing the fermentation solution and vegetables, and fermenting for 3-5 days at 20-25 ℃.
Preferably, the inoculation amount is 0.45-0.5%.
Preferably, in the mixed probiotics, the volume fraction of Lactobacillus plantarum V02 is 30-35%, the volume fraction of Lactobacillus diolivorans V73 is 65-70%, and the bacteria liquid concentration of Lactobacillus plantarum V02 before mixing is (1.6-2.5) x 109CFU/mL, wherein the concentration of the bacterial liquid before mixing of the Lactobacillus diolvorans V73 is (0.8-1.5) multiplied by 109CFU/mL。
Preferably, the preparation method of the pepper water comprises the following steps: mixing 0.3-0.5% of dried pepper, 0.1-0.3% of aniseed, 0.2-0.4% of ginger slices and 98.8-99.4% of water, heating and boiling twice, and cooling.
Preferably, the mass of the pepper water accounts for 80-120% of the mass of the vegetables.
Preferably, the salt accounts for 2.5-3.5% of the weight of the vegetables.
Preferably, the vegetables comprise a combination of lettuce, parsley and carrot; or combination of lettuce, cabbage, parsley and carrot.
Preferably, the vegetables are shredded prior to fermentation.
The invention also provides the fermented vegetable prepared by the method of the technical scheme.
The invention provides a mixed probiotic for fermenting vegetables. According to the invention, Lactobacillus plantarum V02 and Lactobacillus diolvorans V73 are mixed to ferment vegetables, so that not only can the reproduction of mixed bacteria be inhibited, and the product quality be ensured, but also the fermented vegetables obtained by utilizing the mixed probiotics have the function of regulating intestinal flora, prevent cardiovascular diseases and are beneficial to human health, and the artificial inoculation of the fermented vegetables can strengthen the fermentation, reduce the nitrite content, shorten the fermentation period, improve the product quality and reduce the production cost. Test results show that when the mixed probiotics provided by the invention are used for fermenting vegetables, the fermentation time is only 3-5 d, the nitrite content is 0.20-0.27 mg/kg, the product quality is stable, and the production cost is low.
Biological preservation Instructions
Lactobacillus plantarum V02. The strain is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, and the address is No. 3 of West Lu No.1 of Beijing, Chaoyang, China academy of sciences, the preservation number is CGMCC No.16727, and the preservation time is 11 months and 12 days in 2018.
Lactobacillus diolivorans V73. The strain is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, and the address is No. 3 of West Lu No.1 of Beijing, Chaoyang, China academy of sciences, the preservation number is CGMCC No.16728, and the preservation time is 11 months and 12 days in 2018.
Detailed Description
The invention provides mixed probiotics for fermenting vegetables, which consist of Lactobacillus diolvorans V73 and Lactobacillus plantarum V02;
the biological preservation number of the Lactobacillus diolvorans V73 is CGMCC No. 16728;
the biological preservation number of the Lactobacillus plantarum V02 is CGMCC No. 16727.
In the invention, two lactic acid bacteria are separated and screened from traditional naturally fermented vegetables in Shanxi, and have been preserved in China general microbiological culture Collection center in 2018, 11, 12 and the preservation numbers are respectively as follows: CGMCC No.16727 (strain V02), CGMCC No.16728 (strain V73). On an MRS agar culture medium, the colony of the V02 strain is 2-3 mm, the surface of the colony is smooth, the edge is regular, the colony is circular and convex, and the colony is glossy and milk white; individual morphology in short rods, short chain arrangement, G+No Bacillus bacteria were used. The colony of the V73 strain is 1-1.5 mm, the surface of the colony is smooth, the edge is neat, the round shape is slightly convex, and the colony is dull and light white; individual morphology is short rod-like, G+No Bacillus bacteria were used. And (3) performing bacteriostasis spectra on fermentation supernatants of the two strains, wherein V02 has an inhibiting effect on salmonella, Listeria monocytogenes, staphylococcus aureus and enterococcus faecalis, and V73 has an inhibiting effect on salmonella and Listeria monocytogenes.
The preparation method of the two lactobacillus liquid is not particularly limited, and the two lactobacillus liquid can be cultured by adopting a conventional fermentation method well known to the technical personnel in the field. Specifically, in the present invention, the two steps of activating lactic acid bacteria and preparing fermentation broth are preferably as follows:
lactobacillus plantarum (Lactobacillus plantarum) V02: coating a lactobacillus plantarum V02 plate preserved by glycerol at the temperature of-80 ℃, taking a single colony to be transferred into a 250mL conical flask of 50mL MRS liquid culture medium, and culturing for 10 h-14 h on a shaking table at the temperature of 37 ℃ and at the speed of 150 r/min-180 r/min; carrying out amplification culture on V02 to 50ml of MRS liquid culture medium with 2-5% of inoculation amount for 10-12 h.
Lactobacillus (Lactobacillus diolivorans) V73: coating a lactobacillus V73 plate preserved by glycerol at the temperature of-80 ℃, taking a single colony to be transferred into a 250mL conical flask of 50mL MRS liquid culture medium, and culturing for 10 to 14 hours on a shaking table at the temperature of 37 ℃ and at the speed of 150 to 180 r/min; carrying out amplification culture on V73 to 50ml of MRS liquid culture medium with 2-5% of inoculation amount for 22-24 h.
In the invention, the vegetable obtained by combined fermentation of the two strains can maintain the micro-ecological balance of human intestinal flora, improve the immunity of the organism, reduce the number of harmful bacteria, prevent diarrhea, cardiovascular diseases and gastrointestinal diseases and the like.
The invention also provides a vegetable fermentation method based on the mixed probiotics in the technical scheme, which comprises the following steps:
inoculating the mixed probiotics in the technical scheme into a mixed solution of pepper water and salt to obtain a fermentation solution, and mixing the fermentation solution and vegetables, and then preferably fermenting at 20-25 ℃ for 3-5 days, and more preferably fermenting at 22 ℃ for 4 days.
In the present invention, the amount of the inoculation is preferably 0.45 to 0.5%.
In the mixed probiotics, the volume fraction of Lactobacillus plantarum V02 is 30-35%, the volume fraction of Lactobacillus diolivorans V73 is 65-70%, and preferably, the mixing volume ratio of Lactobacillus plantarum V02 to Lactobacillus diolivorans V73 is preferably 1: 2; the concentration of the bacterial liquid before mixing of the Lactobacillus plantarum V02 is (1.6-2.5) multiplied by 109CFU/mL, wherein the concentration of the bacterial liquid before mixing of the Lactobacillus diolvorans V73 is (0.8-1.5) multiplied by 109CFU/mL。
In the invention, the preparation method of the zanthoxylum water preferably comprises the following steps: mixing 0.3-0.5% of dried pepper, 0.1-0.3% of aniseed, 0.2-0.4% of ginger slices and 98.8-99.4% of water, heating and boiling twice, and cooling. The sources of the dried pepper, aniseed and ginger slices are not particularly limited, and conventional commercial products of the conventional dried pepper, aniseed and ginger slices, which are well known to those skilled in the art, can be adopted. In the present invention, the water is preferably purified water. In the invention, the cooling is preferably carried out to be below 30-40 ℃.
In the invention, the mass of the pepper water accounts for 80-120% of the mass of the vegetables, and more preferably, the mixing mass ratio of the pepper water to the vegetables is 1: 1. In the present invention, the salt accounts for 2.5 to 3.5% by mass, more preferably 3.0% by mass of the vegetable.
The kind of the vegetable is not particularly limited in the present invention, and vegetables known to those skilled in the art may be used. In the present invention, the vegetables preferably include lettuce, parsley and carrot; or lettuce, cabbage, parsley and carrot. In the present invention, the vegetables are preferably cut into shreds before fermentation. The fermentation apparatus of the present invention is not particularly limited, and it is preferable to use a conventional container known to those skilled in the art, such as a kimchi jar. In the invention, the fermentation mode is anaerobic fermentation, and the fermentation device is preferably placed in a cool and ventilated place.
The invention also provides the fermented vegetable prepared by the method of the technical scheme. The fermented vegetable obtained by the invention has the functions of regulating intestinal flora, preventing cardiovascular diseases, being beneficial to human health and having high product quality.
The mixed probiotics for fermented vegetables, the fermented vegetables and the preparation method thereof according to the present invention will be further described in detail with reference to the following specific examples, and the technical solutions of the present invention include, but are not limited to, the following examples.
Example 1
Method for making fermented vegetable
(1) 6 lactic acid bacteria V02, V22, V53, V66, V71 and V73 from fermented vegetables are respectively used in vegetable fermentation experiments, three lactic acid bacteria V71, V73 and V02 with better vegetable fermentation effect are screened according to the flavor of the fermented vegetables, and single strain and different lactic acid bacteria are respectively combined in equal proportion to ferment the vegetables.
Sensory evaluation and pH measurement are carried out according to the color, aroma, taste and mouthfeel of the fermented vegetables, an evaluation group is composed of professional evaluators (namely, the evaluators are trained for professional sensory evaluation), 10 persons are in total, the color, aroma, taste and mouthfeel of the samples are respectively and finely described by adopting a descriptive detection method, and the result of the sensory evaluation is used as the evaluation standard of the probiotic fermented vegetables. Sensory evaluation criteria and evaluation indexes of the probiotic fermented vegetables are shown in table 1.
According to the sensory evaluation standard shown in the table 1, the sensory evaluation standard is used as a test index for evaluating the sensory quality of the probiotic fermented vegetables, the sensory evaluation result of the combined fermentation V02 and V73 is best through the sensory evaluation result and pH (the pH value of the acidity end point is less than 3.6), the time for reaching the acidity end point of the fermentation is quicker, and the probiotic fermented vegetables are finally screened for subsequent fermentation.
TABLE 1 sensory evaluation criteria for probiotic fermented vegetables
(2) Culture and viable count of lactic acid bacteria strains
Activating glycerol preserved V02 and V73 at-80 deg.C, performing amplification culture in sterilized MRS liquid culture medium, respectively adding 1mL of bacterial liquid into 9mL sterilized normal saline tube, and making into 10-1Diluting the solution to 10 times in increments-8(ii) a Get 10-6～10-81mL of each diluted solution is placed in a sterile plate, about 15mL of sterilized MRS culture medium which is melted and cooled to 44 ℃ is poured, the plate is quickly and gently swirled, the culture medium and the bacterial liquid are fully mixed, and each dilution is repeated for 3 times. Meanwhile, the sterilized MRS medium was injected into a sterilized dish containing 1mL of sterile physiological saline as a blank control. After the culture medium is solidified, the flat plate is inverted, the flat plate is placed in a temperature box of 36 +/-1 ℃ for culture (48 +/-2) h, and counting can be carried out after bacterial colonies grow out. According to the MRS plate counting result, the number of viable Lactobacillus plantarum V02 is (1.7-2.3) multiplied by 109CFU/ml, V73 viable bacteria of Lactobacillus diolvorans count of (1-1.4) × 109CFU/ml。
(3) Determination of the amount of inoculation of lactic acid bacteria
The two lactic acid bacteria strains are fermented at equal concentration, the inoculation quantity gradient of 0.35 percent, 0.4 percent, 0.45 percent, 0.5 percent and 0.55 percent of the total weight of the vegetables are respectively used in experiments for fermenting the vegetables, and probiotic fermented vegetable products prepared by better inoculation quantity have uniform color, luster, no mildew and stains of the vegetables, strong fragrance, refreshing taste, moderate acidity and salinity, crisp mouthfeel, highest sensory evaluation score and 0.45 to 0.5 percent of the inoculation quantity finally selected according to the evaluation results of the color, the fragrance, the taste and the mouthfeel of the fermented vegetables.
(4) Probiotics fermented vegetable production process
1) The probiotic fermented vegetable production process comprises the following steps: cleaning and shredding fresh lettuce, parsley and carrot (mass ratio is 8:1:1) and putting the fresh lettuce, parsley and carrot into a jar for later use; preparing pepper water (the total weight of vegetables: pepper water is 1:1), putting 0.3-0.5% of dry pepper, 0.1-0.3% of aniseed and 0.2-0.4% of ginger slices and the like into 98.8-99.4% of purified water, heating and boiling twice, and cooling to below 40 ℃ for later use. 2.8 to 3.2 percent (the total weight of the vegetables is taken as percentage) of edible salt is dissolved in pepper water, 0.45 to 0.5 percent of mixed fermentation liquor of two lactic acid bacteria (the volume ratio is V02: V73 is 1:2) is added, the total weight of the vegetables and the mixed pepper water are mixed in equal proportion, the mixture is evenly mixed in a jar, the jar is covered and placed in a shady and ventilated place at 25 ℃ for 3 to 5 days.
2) The preparation method of the formula 2 is characterized in that the raw vegetables in the step 1) are replaced by fresh lettuce, cabbage, parsley and carrot (the mass ratio is 4:4:1:1), and the other methods are consistent.
(5) Total lactobacillus count and pH value in probiotic fermented vegetables
Weighing 25g of fermented vegetables, putting into 225mL of sterilized normal saline, treating for 10s at 8000-10000 r/min by using a slapping homogenizer, and fully oscillating to obtain 10-1To obtain a homogeneous dilution. Then diluted 10 times to 10 times by 9mL of sterilized normal saline-11Take 10-6～10-91mL of each diluted solution is placed in a sterile plate, 15mL of modified MRS culture medium which is dissolved and cooled to 46 ℃ is poured, the plate is quickly and gently swirled, the culture medium and the bacterial liquid are fully mixed, and each dilution is repeated for 3 times. Simultaneously, the improved MRS culture medium is injected into a sterilized plate added with 1mL of sterile physiological saline to be used as a blank control. After the culture medium is solidified, the flat plate is inverted, the flat plate is placed in a temperature box of 36 +/-1 ℃ for culture (48 +/-2) h, and counting can be carried out after bacterial colonies grow out. 50mL of the mixed fermentation broth was taken, and the pH value was measured with a pH meter and recorded. The results of plate colony count and pH value show that the fermentation raw material is lettuce, the number of lactic acid bacteria is (3.5-4.5) x 108CFU/ml, and the pH value is 3.2-3.4 when the fermentation is carried out for 5 days; the fermentation raw materials are lettuce and cabbage, and the number of lactic acid bacteria is (5.5-6.6) x 108CFU/ml, and the pH value is 3.2-3.4 when the fermentation is carried out for 5 days.
(6) Nitrite determination of probiotic fermented vegetables
Weighing 5g (accurate to 0.001g) of homogenate sample, placing the homogenate sample into a 250mL conical flask with a plug, adding 12.5mL of 50g/L saturated borax solution, adding about 150mL of water at about 70 ℃, uniformly mixing, heating in a boiling water bath for 15min, taking out, placing in a cold water bath, cooling, and placing to room temperature. Quantitatively transferring the above extractive solution into a 200mL volumetric flask, adding 5mL106g/L potassium ferrocyanide solution, shaking, and adding 5mL220g/L zinc acetate solution to precipitate protein. Adding water to scale, shaking, standing for 30min, removing upper layer fat, filtering supernatant with filter paper, discarding 30mL of primary filtrate, and reserving filtrate.
Sucking 40.0mL of the filtrate into a 50mL colorimetric tube with a plug, respectively adding 2mL of 4g/L sulfanilic acid solution into a standard tube and a sample tube, uniformly mixing, standing for 3-5 min, respectively adding 1mL of 2g/L naphthyl ethylenediamine hydrochloride solution, adding water to the scale, uniformly mixing, standing for 15min, adjusting the zero point by using a 1cm cuvette by using a zero tube, measuring the absorbance at the position of 538nm wavelength, and drawing a standard curve for comparison. And simultaneously, making a reagent blank. Nitrite standard curve regression equation: y is 0.0477x, R20.9986, the main fermentation raw material is lettuce, and the nitrite content is 0.20-0.27 mg/kg during 5 days of fermentation; the fermentation main raw materials are lettuce and cabbage, and the nitrite content is 0.20-0.27 mg/kg when the fermentation is carried out for 5 days. In the invention, the nitrite content is in the range of 0.10-0.27 mg/kg in the 0 d-5 d fermentation process of inoculated fermented vegetables, no nitrite peak appears in the fermentation process, and the nitrite content is lower and stable; the nitrite content of the naturally fermented vegetables is within the range of 0.10-2.18 mg/kg in the fermentation process of 0 d-5 d, and the nitrite content of the naturally fermented vegetables is higher than that of the inoculated fermented vegetablesWhen the vegetables are naturally fermented for 1d, the nitrite peak reaches 2.18mg/kg, but the content of the nitrite is within a reasonable limit range (the nitrite content of the pickled vegetables is lower than 20mg/kg in the national standard GB 2762-.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (3)
1. A method for vegetable fermentation based on mixed probiotics comprising the steps of:
inoculating the mixed probiotics into a mixed solution of pepper water and salt to obtain a fermentation solution, mixing the fermentation solution with vegetables, and fermenting for 3-5 days at 20-25 ℃;
the mixed probiotics consists ofLactobacillus diolivoransV73 and Lactobacillus plantarumLactobacillus plantarum V02;
the above-mentionedLactobacillus diolivoransThe biological preservation number of V73 is CGMCC No. 16728;
the lactobacillus plantarumLactobacillus plantarum The biological preservation number of V02 is CGMCC No. 16727;
in the mixed probiotics, the lactobacillus plantarumLactobacillus plantarum The volume fraction of V02 is 30-35 percentLactobacillus diolivoransThe volume fraction of V73 is 65-70%, and the lactobacillus plantarumLactobacillus plantarum The concentration of the bacterial liquid before V02 mixing is (1.6-2.5) x 109CFU/mL, saidLactobacillus diolivoransThe concentration of the bacterial liquid before V73 mixing is (0.8-1.5) x 109 CFU/mL；
The inoculation amount is 0.45-0.5%;
the preparation method of the pepper water comprises the following steps: mixing 0.3% -0.5% of dry pepper, 0.1% -0.3% of aniseed, 0.2% -0.4% of ginger slices and 98.8% -99.4% of water, heating and boiling twice, and cooling;
the mass of the pepper water accounts for 80-120% of the mass of the vegetables;
the mass of the salt accounts for 2.5-3.5% of the mass of the vegetables.
2. The method of claim 1, wherein the vegetable is a combination of lettuce, parsley and carrot; or combination of lettuce, cabbage, parsley and carrot.
3. The method of claim 2, wherein the vegetable is shredded prior to fermentation.
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