CN105647808B - A kind of method of agaropectin oligose as protective agent preservation lactobacillus bulgaricus - Google Patents
A kind of method of agaropectin oligose as protective agent preservation lactobacillus bulgaricus Download PDFInfo
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- C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
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Abstract
A kind of method the invention discloses agaropectin oligose as protective agent preservation lactobacillus bulgaricus, comprising the following steps: Step 1: preparing agaropectin oligose；Step 2: Spawn incubation: preparation MRS culture medium, agaropectin oligose are inoculated with lactobacillus bulgaricus OD as carbon source600The bacterium solution that value is 0.8, inoculum concentration 2%, under oxygen-free environment, 42 DEG C, stationary culture 2-3 days；Step 3: liquid bacteria preservation: in 0D600Value is the agaropectin oligose that addition concentration is 1-10g/L and the degree of polymerization is 3-8 in 1.2 lactobacillus bulgaricus liquid, is stored at 4 DEG C；Step 4: solid fungicide preservation: in 0D600Value is in 1.2 lactobacillus bulgaricus liquid, and the agaropectin oligose that addition concentration is 5g/L and the degree of polymerization is 3, spray drying prepares solid fungicide.The present invention has the function of that improving lactobacillus bulgaricus liquid and solid-state microbial inoculum prepares the vigor in preserving process, can be used for lactobacillus-fermented enterprise and dairy products manufacturing enterprise, to produce the dairy products of high-activity lactic acid bacteria product and high storability.
Description
Technical field
The present invention relates to the technical fields of culture presevation more particularly to a kind of agaropectin oligose as protective agent preservation Bao Jiali
The method of sub- lactobacillus.
Background technique
Fragrant plant mentioned in ancient texts (Gracilaria) is perennial large-scale red algae, belongs to Rhodophyta, is China Guangdong, Zhejiang, Taiwan edge
One of seaweeds breed variety, mainly as fishery cultivating feed and production foodstuff glue raw material.Fragrant plant mentioned in ancient texts dish agaropectin oligose is a kind of
Non-digestible oligosaccharide has the effect that is obviously promoted to the growth of lactobacillus bulgaricus, and has and inhibit pathogen, corruption
The growth of the harmful bacterias such as bacterium can improve the immunity of the human body, and improve endocrine system, promote the absorption of nutriment, to human body Asia
Health status is improved, to enteritis caused by flora imbalance caused by antibiotic, hormone, cytotoxic drug etc., diarrhea,
Constipation and side effects of pharmaceutical drugs are treated, and have the function of promoting longevity.Therefore fragrant plant mentioned in ancient texts dish agaropectin oligose is a kind of novel
Health care product and food additives or feed addictive.
Currently, also not having fragrant plant mentioned in ancient texts dish agaropectin oligose to the report of the facilitation effect of probiotics in the prior art, both at home and abroad
The effects of main research the antiviral of oligosaccharides, anti-inflammatory, anticancer., often there is product in industrial production probiotics fermention product
Probiotics quantity problem not up to standard.In view of this, the present inventor's research and devising a kind of agaropectin oligose as protective agent guarantor
Thus the method for hiding lactobacillus bulgaricus, this case generate.
Summary of the invention
A kind of method the purpose of the present invention is to provide agaropectin oligose as protective agent preservation lactobacillus bulgaricus, benefit
It uses fragrant plant mentioned in ancient texts dish agaropectin oligose as the carbon source of lactobacillus bulgaricus, protects work of the lactobacillus bulgaricus under cryopreservation
Property, lactobacillus bulgaricus survival rate in human stomach intestinal juice is protected, the bacterium vigor of lactobacillus bulgaricus is improved.
To achieve the goals above, the technical scheme adopted by the invention to solve the technical problem is that:
A kind of method of agaropectin oligose as protective agent preservation lactobacillus bulgaricus, comprising the following steps:
Step 1: preparing agaropectin oligose: fragrant plant mentioned in ancient texts being cleaned and is cleaned, drying, 30 times of fragrant plant mentioned in ancient texts weight of water, which is added, fills fragrant plant mentioned in ancient texts
Divide swelling, is placed in 80 DEG C of water-bath and heats 2h；High pressure sterilization, 121 DEG C of sterilizing 20min；By fragrant plant mentioned in ancient texts plastic squeeze while hot, with filter cloth packet
Fragrant plant mentioned in ancient texts is wrapped up in, and is squeezed using press machine, the polysaccharide solution that plastic squeeze filters out is collected, cools down, dries, being ground into particle to get fragrant plant mentioned in ancient texts
Thick many candies；By in cooling Thick many candies dissolution water, the polysaccharide solution that concentration is 5g/L is made, agar-agar enzymatic treatment is added；Enzyme concentration
For 15% (v/v), enzyme activity 300U/mL；Digest 20-1280min, to prepare oligosaccharide solution, freeze-drying, or vacuum drying at
The agaropectin oligose that the degree of polymerization is 3-8 is made in solid powder；
Step 2: Spawn incubation: preparation MRS culture medium, agaropectin oligose are inoculated with lactobacillus bulgaricus OD as carbon source600
The bacterium solution that value is 0.8, inoculum concentration 2%, under oxygen-free environment, 42 DEG C, stationary culture 2-3 days；
Step 3: liquid bacteria preservation: in 0D600Value is in 1.2 lactobacillus bulgaricus liquid, and addition concentration is 1-10g/L
And the degree of polymerization is the agaropectin oligose of 3-8, places and stores at 4 DEG C；
Step 4: solid fungicide preservation: in 0D600Value is in 1.2 lactobacillus bulgaricus liquid, and addition concentration is 5g/L
And the agaropectin oligose that the degree of polymerization is 3, spray drying prepare solid fungicide, spray drying condition are as follows: maltodextrin additive amount 25%,
130 DEG C of inlet air temperature, intake velocity 4m3/ min, charging rate 700mL/h, air pressure 0.3Pa prepare solid fungicide, preparation
Good solid fungicide is placed on room temperature or -20 DEG C of storages.
As the preferred embodiment of embodiment, in the step 1, the agar-agar that the degree of polymerization is 8 is made in enzymolysis time 20min
Oligosaccharides.
As the preferred embodiment of embodiment, in the step 1, the agar-agar that the degree of polymerization is 5 is made in enzymolysis time 80min
Oligosaccharides.
As the preferred embodiment of embodiment, in the step 1, the fine jade that the degree of polymerization is 3 is made in enzymolysis time 1280min
Glue oligosaccharides.
As the preferred embodiment of embodiment, in the step 2, the composition of MRS culture medium are as follows: beef protein powder 10g, fish
Gravy 10g, Yeast diffusion juice powder 5g, agaropectin oligose 20g, sodium acetate 5g, dibasic ammonium citrate 2g, Tween 80 0.1g, magnesium sulfate
0.58g, manganese sulfate 0.28g, aquae destillata 1000ml；121 DEG C of high pressure sterilization 15min adjust pH6.2-6.4.
After technical solution of the present invention, have the advantages that
1. the present invention effectively increases guarantor and adds using fragrant plant mentioned in ancient texts dish agaropectin oligose as the growth carbon source of lactobacillus bulgaricus
Activity of the Leah lactobacillus under cryopreservation and under bacteria preparation and the protective effect under gastro-intestinal digestion environment.
2. fragrant plant mentioned in ancient texts dish agaropectin oligose used in the present invention will not generate any pollutant component, do not pollute the environment more.This
Small investment is quick in actual production for method, is suitble to promote and apply.
3. being heated using high pressure sterilization, effectively fragrant plant mentioned in ancient texts dish can be heated, when reducing heating water bath
Between, reduce heat energy loss, the call of respective country energy-saving and emission-reduction.
Detailed description of the invention
Fig. 1 is effect of the different polymerization degree agaropectin oligose of the present invention to lactobacillus bulgaricus in solid-state microbial inoculum；
Fig. 2 is effect of the various concentration agaropectin oligose of the present invention to lactobacillus bulgaricus in solid-state microbial inoculum；
Fig. 3 is that agaropectin oligose of the present invention improves tolerance effect of the lactobacillus bulgaricus in gastric juice simulated solution；
Fig. 4 is that agaropectin oligose of the present invention improves tolerance effect of the lactobacillus bulgaricus in intestinal juice simulated solution.
Specific embodiment
Enzyme-activity unit is defined as enzyme amount needed for generating 1 μ g reduced sugar per minute.
Blank control group: preparation MRS culture medium, the composition of MRS culture medium are as follows: beef protein powder 10g, flesh of fish juice 10g, ferment
Female diffusion juice powder 5g, glucose 20g, sodium acetate 5g, dibasic ammonium citrate 2g, Tween 80 0.1g, magnesium sulfate 0.58g, manganese sulfate
0.28g, aquae destillata 1000ml；121 DEG C of high pressure sterilization 15min adjust pH6.2-6.4；Glucose is inoculated with Bao Jiali as carbon source
Sub- lactobacillus OD600The bacterium solution that value is 0.8, inoculum concentration 2%.Under oxygen-free environment, 42 DEG C, stationary culture 2-3 days.
Fragrant plant mentioned in ancient texts dish agaropectin oligose group: agaropectin oligose is replaced the grape in MRS culture medium by preparation fragrant plant mentioned in ancient texts dish agaropectin oligose
Sugar, and utilize the oligosaccharides (DP3, DP5, DP8) and various concentration oligosaccharides (1g/L, 3g/L, 5g/L, 7g/L, 10g/ of different polymerization degree
L) lactobacillus bulgaricus cultivated.It is inoculated with lactobacillus bulgaricus OD600The bacterium solution that value is 0.8, inoculum concentration 2%.
Under oxygen-free environment, 42 DEG C, stationary culture 2-3 days.
A kind of embodiment 1: fragrant plant mentioned in ancient texts dish agaropectin oligose preparation method of lactobacillus bulgaricus growth
Step 1: fragrant plant mentioned in ancient texts cleans: fragrant plant mentioned in ancient texts being cleaned and is cleaned, until fragrant plant mentioned in ancient texts weight is added in no silt, water clear, drying
30 times of water is swollen fragrant plant mentioned in ancient texts sufficiently, is subsequently placed in 80 DEG C of water-bath and heats 2h；
Step 2: high pressure sterilization: high pressure sterilization is carried out to the fragrant plant mentioned in ancient texts after heat treatment, by microorganism killing, high pressure sterilization
Temperature is 121 DEG C, sterilization time 20min；
Step 3: plastic squeeze: by fragrant plant mentioned in ancient texts plastic squeeze while hot, wrapping up fragrant plant mentioned in ancient texts, and the filter using press machine to fragrant plant mentioned in ancient texts is equipped with filter cloth
Cloth is squeezed, and the polysaccharide solution that plastic squeeze filters out is collected；The polysaccharide solution of collection cool down, is dried, is ground into particle dress
Bag is spare, is placed at shady and cool drying and saves to get fragrant plant mentioned in ancient texts Thick many candies；
Step 4: enzymatic treatment: by cooling Thick many candies dissolution water, the polysaccharide solution that concentration is 5g/L is made, fine jade is added
Glue enzymatic treatment；Enzyme concentration is 15% (v/v), enzyme activity 300U/mL；20-1280min is digested, to prepare oligosaccharide solution；
Step 5: dry: oligosaccharide solution is freeze-dried, or agaropectin oligose is made at solid powder in vacuum drying.
As the preferred embodiment of embodiment 1, in the step 4, the fine jade that the degree of polymerization is 8 is made in enzymolysis time 20min
Glue oligosaccharides.
As the preferred embodiment of embodiment 1, in the step 4, the fine jade that the degree of polymerization is 5 is made in enzymolysis time 80min
Glue oligosaccharides.
As the preferred embodiment of embodiment 1, in the step 4, enzymolysis time 1280min, it is 3 that the degree of polymerization, which is made,
Agaropectin oligose.
Embodiment 2: different polymerization degree agaropectin oligose is to the shadow for improving lactobacillus bulgaricus bacterium vigor under cryopreservation
It rings
The agaropectin oligose that different polymerization degree is prepared using oligosaccharides method made above, is added it to have fermented and reaches
OD600In MRS culture medium of the value for 0.8 lactobacillus bulgaricus, according to the different polymerization degree agaropectin oligose of same concentration
(DP3, DP5, DP8) is added in culture medium, and condition of culture is 4 DEG C under oxygen-free environment, and stationary culture was sampled every 3 days to aobvious
Micro- microscopic observation number of viable, as shown in table 1.
1 different polymerization degree agaropectin oligose of table improves lactobacillus bulgaricus bacterium vigor under cryopreservation
The result shows that under conditions of fragrant plant mentioned in ancient texts dish agaropectin oligose (DP3), to lactobacillus bulgaricus under cryopreservation
Protective effect is best.
Embodiment 3: various concentration gracilaria verrucosa agar gel oligosaccharides is to raising lactobacillus bulgaricus bacterium vigor under cryopreservation
It influences
The agaropectin oligose that various concentration is prepared using oligosaccharides method made above, is added it to have fermented and reaches OD600
In MRS culture medium of the value for 1.2 lactobacillus bulgaricus, by the agaropectin oligose of the various concentration of the degree of polymerization 3 (1g/L, 3g/L,
5g/L, 7g/L, 10g/L) it is added in culture medium, condition of culture is 4 DEG C under oxygen-free environment, and stationary culture was sampled every 3 days
To microscopically observation number of viable, as shown in table 2.
2 various concentration gracilaria verrucosa agar gel oligosaccharides of table improves lactobacillus bulgaricus bacterium vigor under cryopreservation
The result shows that being store to lactobacillus bulgaricus in low temperature under the conditions of fragrant plant mentioned in ancient texts dish agaropectin oligose concentration is 1-10g/L
Hiding is lower to have protective effect, and under conditions of 5g/L, it is best to protective effect of the lactobacillus bulgaricus under cryopreservation.
Embodiment 4: effect of the different polymerization degree agaropectin oligose to lactobacillus bulgaricus in solid-state microbial inoculum preparation
It is added to have fermented using the agaropectin oligose that oligosaccharides method made above prepares different polymerization degree and reaches OD600Value
In bacterium solution for 1.2 lactobacillus bulgaricus, the different polymerization degree agaropectin oligose (DP3, DP5, DP8) that concentration is 5g/L is added
It adds in mixed liquor, by preparing solid-state microbial inoculum using spray dryer, spray dryer running parameter is maltodextrin addition
Amount 25%, 130 DEG C of inlet air temperature, intake velocity 4m3/ min, feed rate 700mL/h, air pressure 0.3MPa.Weigh preparation
Solid-state microbial inoculum 1g be coated plate measurement viable count, as shown in Figure 1.
The result shows that degree of polymerization 3-8 is effective in the preparation of raising solid-state microbial inoculum bacterium vigor, but the optimal oligosaccharides degree of polymerization
It is 3.
Embodiment 5: effect of the various concentration agaropectin oligose to lactobacillus bulgaricus in solid-state microbial inoculum preparation
It is added to have fermented using the agaropectin oligose that oligosaccharides method made above prepares various concentration and reaches OD600Value is
In the bacterium solution of 1.2 lactobacillus bulgaricus, the various concentration agaropectin oligose (1g/L-10g/L) that the degree of polymerization is 5 is added to mixed
It closes in liquid, by preparing solid-state microbial inoculum using spray dryer, spray dryer running parameter is maltodextrin additive amount 25%,
130 DEG C of inlet air temperature, intake velocity 4m3/ min, feed rate 700mL/h, air pressure 0.3MPa.Weigh the solid-state bacterium of preparation
Agent 1g is coated plate measurement viable count, as shown in Figure 2.
The result shows that the effective optimal oligosaccharide concentration of concentration 1-10g/L is in the preparation of raising solid-state microbial inoculum bacterium vigor
5g/L。
Embodiment 6: agaropectin oligose improves lactobacillus bulgaricus tolerance in stomach and intestine simulated solution and acts on process flow
The agaropectin oligose that degree of polymerization 3-8 is prepared using oligosaccharides method made above, reaches OD for having fermented600Value is 1.2
The bacterium solution of lactobacillus bulgaricus mixed with various concentration agaropectin oligose, then mixed with gastro-intestinal Fluid by 1:1 (v/v), cultivate item
Part is 37 DEG C of heating in water bath for reaction 3h, and stationary culture is diluted spread plate, plate culture item every 45min sampling when from 0
Part is under oxygen-free environment, 42 DEG C bio-incubator static gas wave refrigerator 2 days, number colony counts, optimal conditions is as shown in Figures 3 and 4.
The result shows that improving optimal oligosaccharides additive amount in alimentary canal tolerance sexuality is that the degree of polymerization is 3 and concentration is 5g/L.
In the present invention, average degree of polymerization 3-8 and concentration 1-10g/L fragrant plant mentioned in ancient texts dish agaropectin oligose, which have, improves bulgarian milk
The effect of bacillus liquid and solid-state microbial inoculum the preparation vigor in preserving process.The present invention utilizes fragrant plant mentioned in ancient texts dish agaropectin oligose to benefit for the first time
Raw bacterium culture ingredient is added or replaces, and so that lactobacillus bulgaricus is slowed down the death of thallus under cryopreservation, improves
The activity of lactobacillus bulgaricus.The present invention extends the preservation time of lactobacillus bulgaricus, and agaropectin oligose is as benefit
Raw member greatly enhances protective effect of the lactobacillus bulgaricus under low temperature, digestive environments, to improve product probiotics
Quality and economic value.Those skilled in the art directly can export or associate from the disclosure of invention and is all
Deformation, is considered as protection scope of the present invention.
Claims (5)
1. a kind of method of agaropectin oligose as protective agent preservation lactobacillus bulgaricus, it is characterised in that: the following steps are included:
Step 1: preparing agaropectin oligose: fragrant plant mentioned in ancient texts being cleaned and is cleaned, drying, 30 times of fragrant plant mentioned in ancient texts weight of water, which is added, keeps fragrant plant mentioned in ancient texts sufficiently molten
It is swollen, it is placed in 80 DEG C of water-bath and heats 2h；High pressure sterilization, 121 DEG C of sterilizing 20min；By fragrant plant mentioned in ancient texts plastic squeeze while hot, river is wrapped up with filter cloth
Li, and utilization press machine squeezes, and collects the polysaccharide solution that plastic squeeze filters out, and cools down, dries, it is slightly more to get fragrant plant mentioned in ancient texts to be ground into particle
Sugar；By in cooling Thick many candies dissolution water, the polysaccharide solution that concentration is 5g/L is made, agar-agar enzymatic treatment is added；Enzyme concentration is
15% (v/v), enzyme activity 300U/mL；20-1280min is digested, to prepare oligosaccharide solution, freeze-drying, or vacuum drying is at solid
The agaropectin oligose that the degree of polymerization is 3-8 is made in body powder；
Step 2: Spawn incubation: preparation MRS culture medium, agaropectin oligose are inoculated with lactobacillus bulgaricus OD as carbon source600Value is
0.8 bacterium solution, inoculum concentration 2%, under oxygen-free environment, 42 DEG C, stationary culture 2-3 days；
Step 3: liquid bacteria preservation: in 0D600Value is in 1.2 lactobacillus bulgaricus liquid, and addition concentration is 1-10g/L and gathers
The right agaropectin oligose for 3-8 is placed and is stored at 4 DEG C；
Step 4: solid fungicide preservation: in 0D600Value is in 1.2 lactobacillus bulgaricus liquid, and addition concentration is 5g/L and gathers
The right agaropectin oligose for being 3, spray drying prepare solid fungicide, spray drying condition are as follows: maltodextrin additive amount 25%, air inlet
130 DEG C of temperature, intake velocity 4m3/ min, charging rate 700mL/h, air pressure 0.3Pa prepare solid fungicide, prepare
Solid fungicide is placed on room temperature or -20 DEG C of storages.
2. a kind of method of the agaropectin oligose as described in claim 1 as protective agent preservation lactobacillus bulgaricus, feature
Be: in the step 1, the agaropectin oligose that the degree of polymerization is 8 is made in enzymolysis time 20min.
3. a kind of method of the agaropectin oligose as described in claim 1 as protective agent preservation lactobacillus bulgaricus, feature
Be: in the step 1, the agaropectin oligose that the degree of polymerization is 5 is made in enzymolysis time 80min.
4. a kind of method of the agaropectin oligose as described in claim 1 as protective agent preservation lactobacillus bulgaricus, feature
Be: in the step 1, the agaropectin oligose that the degree of polymerization is 3 is made in enzymolysis time 1280min.
5. a kind of method of the agaropectin oligose as described in claim 1 as protective agent preservation lactobacillus bulgaricus, feature
It is: in the step 2, the composition of MRS culture medium are as follows: beef protein powder 10g, flesh of fish juice 10g, Yeast diffusion juice powder 5g, fine jade
Glue oligosaccharides 20g, sodium acetate 5g, dibasic ammonium citrate 2g, Tween 80 0.1g, magnesium sulfate 0.58g, manganese sulfate 0.28g, aquae destillata
1000ml；121 DEG C of high pressure sterilization 15min adjust pH6.2-6.4.
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| CN106260546A (en) * | 2016-10-06 | 2017-01-04 | 集美大学 | A kind of preparation method of enzymolysis Gracilaria tenuistipitata lactic acid bacteria microgranule feedstuff |
| CN108315279A (en) * | 2018-04-09 | 2018-07-24 | 河南科技大学 | A kind of long-time storage method of lactobacillus plantarum liquid preparation |
| CN113817710B (en) * | 2021-11-09 | 2023-11-24 | 蓝脑科技(厦门)有限公司 | Agarase freeze-drying protective agent and agarase preservation method |
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Denomination of invention: Method for preserving Lactobacillus bulgaricus with agar oligosaccharide as protective agent Effective date of registration: 20210729 Granted publication date: 20190927 Pledgee: Bank of Xiamen Limited by Share Ltd. Zhangzhou branch Pledgor: FUJIAN PROVINCE LVQI FOOD COLLOID Co.,Ltd. Registration number: Y2021350000082 |
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