CN105506032A - Preparation method and application of Gracilaria agar oligosaccharide promoting growth of Lactobacillus bulgaricus - Google Patents
Preparation method and application of Gracilaria agar oligosaccharide promoting growth of Lactobacillus bulgaricus Download PDFInfo
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Abstract
The invention discloses a preparation method of Gracilaria agar oligosaccharide promoting growth of Lactobacillus bulgaricus. The preparation method includes following steps: step 1, removing impurities of Gracilaria: cleaning, removing impurities of Gracilaria, drying, and placing Gracilaria in water bath at 80 DEG C for heating for 2 h; step 2, sterilizing at high temperature: sterilizing at 121 DEG C for 20 min; step 3, squeezing glue: using filter cloth to wrap Gracilaria, squeezing, collecting a polysaccharide solution, cooling, drying, smashing into granules, and bagging the granules for standby use; step 4, performing enzymatic treatment: dissolving cooled coarse polysaccharide in water to prepare a polysaccharide solution of 5g/L in concentration, adding agarase for treatment, performing enzymolysis for 20-1280 min to prepare an oligosaccharide solution, wherein enzyme adding quantity is 15% and enzyme activity is 300 U/mL; step 5, drying: freeze-drying the oligosaccharide solution or vacuum-drying the same to form solid powder to obtain the agar oligosaccharide. The preparation method can be used for Lactobacillus fermentation enterprises and dairy product production enterprises to produce high-activity Lactobacillus products and high-storability dairy products.
Description
Technical field
The present invention relates to the technical field of food-processing, particularly relate to a kind of fragrant plant mentioned in ancient texts dish agaropectin oligose preparation method and application thereof of short lactobacillus bulgaricus growth.
Background technology
Fragrant plant mentioned in ancient texts (Gracilaria) is perennial large-scale red algae, belongs to rhodophyta, is one of China Guangdong, Zhejiang, the coastal algal culture kind in Taiwan, 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, to the growth of lactobacillus bulgaricus, there is obvious promoter action, and there is the growth suppressing the harmful bacteria such as pathogenic bacteria, spoilage organism, body immunity can be improved, improve endocrine system, promote the absorption of nutritive substance, human body sub-health status is improved, enteritis caused by the flora imbalance caused to microbiotic, hormone, cytotoxic drug etc., diarrhoea, constipation and Side effects of pharmaceutical drugs are treated, and have the effect of promoting longevity.Therefore fragrant plant mentioned in ancient texts dish agaropectin oligose is a kind of new health care product and foodstuff additive or fodder additives.
At present, also there is not fragrant plant mentioned in ancient texts dish agaropectin oligose in the prior art to the report of the facilitation effect of probiotic bacterium, both at home and abroad the effect such as antiviral, anti-inflammatory, anticancer of main research oligosaccharides., often there is the problem that product probiotic bacterium quantity is not up to standard in industrial production probiotics fermention product.In view of this, the present inventor studies and devises a kind of fragrant plant mentioned in ancient texts dish agaropectin oligose preparation method and application thereof of short lactobacillus bulgaricus growth, and this case produces thus.
Summary of the invention
The object of the present invention is to provide a kind of fragrant plant mentioned in ancient texts dish agaropectin oligose preparation method and application thereof of short lactobacillus bulgaricus growth, utilize fragrant plant mentioned in ancient texts dish agaropectin oligose as the carbon source of lactobacillus bulgaricus, improve fermentation efficiency, reduce factory's fermentation time, promote lactobacillus bulgaricus growth.
To achieve these goals, the present invention solves the technical scheme that its technical problem takes and is:
A fragrant plant mentioned in ancient texts dish agaropectin oligose preparation method for short lactobacillus bulgaricus growth, comprises the following steps:
Step one, fragrant plant mentioned in ancient texts removal of impurities: fragrant plant mentioned in ancient texts is cleaned removal of impurities, dry, the water adding fragrant plant mentioned in ancient texts weight 30 times makes fragrant plant mentioned in ancient texts fully swelling, and 2h is heated in the water-bath being then placed in 80 DEG C;
Step 2, autoclaving: autoclaving is carried out to the fragrant plant mentioned in ancient texts after heat treated, by microorganism killing, autoclave temperature is 121 DEG C, and sterilization time is 20min;
Step 3, plastic squeeze: by fragrant plant mentioned in ancient texts plastic squeeze while hot, wrap up fragrant plant mentioned in ancient texts with filter cloth, and utilize pressing machine to extrude the filter cloth that fragrant plant mentioned in ancient texts is housed, collect the polysaccharide soln that plastic squeeze leaches; The polysaccharide soln of collection carried out cool, dry, to be ground into particle pack for subsequent use, be placed on shady and cool dry place and preserve, obtain fragrant plant mentioned in ancient texts Crude polysaccharides;
Step 4, ferment treatment: by the Crude polysaccharides dissolved water of cooling, make the polysaccharide soln that concentration is 5g/L, add agarase process; Enzyme concentration is 15% (v/v), and enzyme is lived as 300U/mL; Enzymolysis 20-1280min, to prepare oligosaccharide solution;
Step 5, drying: oligosaccharide solution carries out lyophilize, or vacuum-drying becomes pressed powder, obtained agaropectin oligose.
As the optimal way of embodiment, in described step 4, enzymolysis time is 20min, and the obtained polymerization degree is the agaropectin oligose of 8.
As the optimal way of embodiment, in described step 4, enzymolysis time is 80min, and the obtained polymerization degree is the agaropectin oligose of 5.
As the optimal way of embodiment, in described step 4, enzymolysis time is 1280min, and the obtained polymerization degree is the agaropectin oligose of 3.
A kind of application of fragrant plant mentioned in ancient texts dish agaropectin oligose of short lactobacillus bulgaricus growth, for low temperature storage lactobacillus bulgaricus: by lactobacillus bulgaricus by inoculum size 2%, being added into containing polymerization degree 3-8 and concentration is in the substratum of the agaropectin oligose of 1-10g/L, be placed in 42 DEG C, under oxygen free condition, quiescent culture 2-3 days, and measure viable count at set intervals.
A kind of application of fragrant plant mentioned in ancient texts dish agaropectin oligose of short lactobacillus bulgaricus growth, for the preparation of the solid-state microbial inoculum of lactobacillus bulgaricus: fermentation lactobacillus bulgaricus is to a certain degree added agaropectin oligose and prepares solid-state microbial inoculum by spraying dry, and measure viable count.
As the optimal way of embodiment, the spray parameters that described spraying dry prepares solid-state microbial inoculum is maltodextrin addition 25%, inlet temperature 130 DEG C, intake velocity 4m
3/ min, feeding rate 700mL/h, air pressure 0.3MPa.
A kind of application of fragrant plant mentioned in ancient texts dish agaropectin oligose of short lactobacillus bulgaricus growth, for the effect of simulating gastrointestinal digestion to lactobacillus bulgaricus: fermentation lactobacillus bulgaricus is to a certain degree added agaropectin oligose and is positioned in in-vitro simulated human body intestinal juice or simulation human gastric juice, measure the tolerance of lactobacillus bulgaricus.
As the optimal way of embodiment, described in-vitro simulated human body intestinal juice condition is: KH
2pO
40.5%, trypsinase 0.3%, regulates pH to be 8.0,0.22 μm of filtering with microporous membrane sterilizing with NaOH.
As the optimal way of embodiment, described in-vitro simulated human gastric juice condition is: NaCl0.3%, KCl0.2%, stomach en-0.5%, regulates pH to be 2.0,0.22 μm of filtering with microporous membrane sterilizing with HCl.
After adopting technical scheme of the present invention, there is following beneficial effect:
1. the present invention utilizes fragrant plant mentioned in ancient texts dish agaropectin oligose as the growth carbon source of lactobacillus bulgaricus, effectively improves the activity of lactobacillus bulgaricus under low temperature storage and under bacteria preparation and the provide protection under gastro-intestinal digestion environment.
2. fragrant plant mentioned in ancient texts dish agaropectin oligose used in the present invention can not produce any pollutant component, does not more pollute the environment.Present method is less investment instant effect in actual production, is applicable to applying.
3. adopt autoclaving heat treated, effectively can carry out heat treated to fragrant plant mentioned in ancient texts dish, reduce the heating in water bath time, reduce heat energy loss, the call of respective country energy-saving and emission-reduction.
Accompanying drawing explanation
Fig. 1 is that different polymerization degree agaropectin oligose of the present invention is to the effect of lactobacillus bulgaricus in solid-state microbial inoculum;
Fig. 2 is that different concns agaropectin oligose of the present invention is to the effect of lactobacillus bulgaricus in solid-state microbial inoculum;
Fig. 3 is that agaropectin oligose of the present invention improves the tolerance effect of lactobacillus bulgaricus in gastric juice simulated solution;
Fig. 4 is that agaropectin oligose of the present invention improves the tolerance effect of lactobacillus bulgaricus in intestinal juice simulated solution.
Embodiment
Enzyme unit definition of living produces enzyme amount needed for 1 μ g reducing sugar for per minute.
Blank group: preparation MRS substratum, glucose, as carbon source, inoculates lactobacillus bulgaricus OD
600value is the bacterium liquid of 0.8, and inoculum size is 2%.Under oxygen-free environment, 42 DEG C, quiescent culture 2-3 days.
Fragrant plant mentioned in ancient texts dish agaropectin oligose group: preparation fragrant plant mentioned in ancient texts dish agaropectin oligose, agaropectin oligose is replaced the glucose in MRS substratum, and utilize the oligosaccharides of different polymerization degree (DP3, DP5, DP8) and different concns oligosaccharides (1g/L, 3g/L, 5g/L, 7g/L, 10g/L) to cultivate lactobacillus bulgaricus.Inoculation lactobacillus bulgaricus OD
600value is the bacterium liquid of 0.8, and inoculum size is 2%.Under oxygen-free environment, 42 DEG C, quiescent culture 2-3 days.
Embodiment 1: a kind of fragrant plant mentioned in ancient texts dish agaropectin oligose preparation method of lactobacillus bulgaricus growth
Step one, fragrant plant mentioned in ancient texts removal of impurities: fragrant plant mentioned in ancient texts is cleaned removal of impurities, to there is no silt, water clear, dry, the water adding fragrant plant mentioned in ancient texts weight 30 times makes fragrant plant mentioned in ancient texts fully swelling, and 2h is heated in the water-bath being then placed in 80 DEG C;
Step 2, autoclaving: autoclaving is carried out to the fragrant plant mentioned in ancient texts after heat treated, by microorganism killing, autoclave temperature is 121 DEG C, and sterilization time is 20min;
Step 3, plastic squeeze: by fragrant plant mentioned in ancient texts plastic squeeze while hot, wrap up fragrant plant mentioned in ancient texts with filter cloth, and utilize pressing machine to extrude the filter cloth that fragrant plant mentioned in ancient texts is housed, collect the polysaccharide soln that plastic squeeze leaches; The polysaccharide soln of collection carried out cool, dry, to be ground into particle pack for subsequent use, be placed on shady and cool dry place and preserve, obtain fragrant plant mentioned in ancient texts Crude polysaccharides;
Step 4, ferment treatment: by the Crude polysaccharides dissolved water of cooling, make the polysaccharide soln that concentration is 5g/L, add agarase process; Enzyme concentration is 15% (v/v), and enzyme is lived as 300U/mL; Enzymolysis 20-1280min, to prepare oligosaccharide solution;
Step 5, drying: oligosaccharide solution carries out lyophilize, or vacuum-drying becomes pressed powder, obtained agaropectin oligose.
As the optimal way of embodiment 1, in described step 4, enzymolysis time is 20min, and the obtained polymerization degree is the agaropectin oligose of 8.
As the optimal way of embodiment 1, in described step 4, enzymolysis time is 80min, and the obtained polymerization degree is the agaropectin oligose of 5.
As the optimal way of embodiment 1, in described step 4, enzymolysis time is 1280min, and the obtained polymerization degree is the agaropectin oligose of 3.
Embodiment 2: different polymerization degree agaropectin oligose improves the technical process of lactobacillus bulgaricus bacterium vigor under low temperature storage
Prepare the agaropectin oligose that oligosaccharides method prepares different polymerization degree more than utilizing, being added into ferments reaches OD
600value is in the MRS substratum of the lactobacillus bulgaricus of 0.8, be added in substratum according to the different polymerization degree agaropectin oligose (DP3, DP5, DP8) of same concentration, culture condition is under oxygen-free environment, 4 DEG C, quiescent culture, basis of microscopic observation number of viable is sampled to every 3 days, as shown in table 1.
Table 1 different polymerization degree agaropectin oligose improves lactobacillus bulgaricus bacterium vigor under low temperature storage
Result shows, under the condition of fragrant plant mentioned in ancient texts dish agaropectin oligose (DP3), best to the provide protection of lactobacillus bulgaricus under low temperature storage.
Embodiment 3: different concns gracilaria verrucosa agar gel oligosaccharides improves the technical process of lactobacillus bulgaricus bacterium vigor under low temperature storage
Prepare the agaropectin oligose that oligosaccharides method prepares different concns more than utilizing, being added into ferments reaches OD
600value is in the MRS substratum of the lactobacillus bulgaricus of 1.2, the agaropectin oligose (1g/L, 3g/L, 5g/L, 7g/L, 10g/L) of the different concns of the polymerization degree 3 is added in substratum, culture condition is under oxygen-free environment, 4 DEG C, quiescent culture, basis of microscopic observation number of viable is sampled to every 3 days, as shown in table 2.
Table 2 different concns gracilaria verrucosa agar gel oligosaccharides improves lactobacillus bulgaricus bacterium vigor under low temperature storage
Result shows, under fragrant plant mentioned in ancient texts dish agaropectin oligose concentration is 1-10g/L condition, has provide protection to lactobacillus bulgaricus under low temperature storage, and under the condition of 5g/L, best to the provide protection of lactobacillus bulgaricus under low temperature storage.
Embodiment 4: different polymerization degree agaropectin oligose is to the effect technical process of lactobacillus bulgaricus in solid-state microbial inoculum preparation
Utilize and above prepare agaropectin oligose that oligosaccharides method prepares different polymerization degree and be added into ferment and reach OD
600value is in the bacterium liquid of the lactobacillus bulgaricus of 1.2, be that the different polymerization degree agaropectin oligose (DP3, DP5, DP8) of 5g/L is added in mixed solution by concentration, solid-state microbial inoculum is prepared by utilizing spray-drier, spray-drier working parameter is maltodextrin addition 25%, inlet temperature 130 DEG C, intake velocity 4m
3/ min, feeding rate 700mL/h, air pressure 0.3MPa.The solid-state microbial inoculum 1g taking preparation carries out spread plate and measures viable count, as shown in Figure 1.
Result shows, improve polymerization degree 3-8 in the preparation of solid-state microbial inoculum bacterium vigor all effective, but the optimum oligosaccharides polymerization degree is 3.
Embodiment 5: different concns agaropectin oligose is to the effect technical process of lactobacillus bulgaricus in solid-state microbial inoculum preparation
Utilize and above prepare agaropectin oligose that oligosaccharides method prepares different concns and be added into ferment and reach OD
600value is in the bacterium liquid of the lactobacillus bulgaricus of 1.2, by the polymerization degree be 5 different concns agaropectin oligose (1g/L-10g/L) be added in mixed solution, solid-state microbial inoculum is prepared by utilizing spray-drier, spray-drier working parameter is maltodextrin addition 25%, inlet temperature 130 DEG C, intake velocity 4m
3/ min, feeding rate 700mL/h, air pressure 0.3MPa.The solid-state microbial inoculum 1g taking preparation carries out spread plate and measures viable count, as shown in Figure 2.
Result shows, improving all effective optimum oligosaccharide concentration of concentration 1-10g/L in the preparation of solid-state microbial inoculum bacterium vigor is 5g/L.
Embodiment 6: agaropectin oligose improves lactobacillus bulgaricus tolerance effect technical process in stomach and intestine simulated solution
Preparing the agaropectin oligose that oligosaccharides method prepares polymerization degree 3-8 more than utilizing, reaching OD by fermenting
600value is that the bacterium liquid of the lactobacillus bulgaricus of 1.2 mixes with different concns agaropectin oligose, mix by 1:1 (v/v) with gastrointestinal fluid again, culture condition is 37 DEG C of heating in water bath for reaction 3h, quiescent culture, from 0 time carry out dilution spread flat board every 45min sampling, slat chain conveyor condition was under oxygen-free environment, 42 DEG C of bio-incubator static gas wave refrigerator 2 days, number colony counts, optimal conditions as shown in Figures 3 and 4.
Result shows, to improve optimum oligosaccharides addition in digestive tube tolerance ability be the polymerization degree be 3 and concentration be 5g/L.
All distortion that those of ordinary skill in the art can directly derive from the disclosure of invention or associate, all should think protection scope of the present invention.
Claims (10)
1. a fragrant plant mentioned in ancient texts dish agaropectin oligose preparation method for short lactobacillus bulgaricus growth, is characterized in that: comprise the following steps:
Step one, fragrant plant mentioned in ancient texts removal of impurities: fragrant plant mentioned in ancient texts is cleaned removal of impurities, dry, the water adding fragrant plant mentioned in ancient texts weight 30 times makes fragrant plant mentioned in ancient texts fully swelling, and 2h is heated in the water-bath being then placed in 80 DEG C;
Step 2, autoclaving: autoclaving is carried out to the fragrant plant mentioned in ancient texts after heat treated, by microorganism killing, autoclave temperature is 121 DEG C, and sterilization time is 20min;
Step 3, plastic squeeze: by fragrant plant mentioned in ancient texts plastic squeeze while hot, wrap up fragrant plant mentioned in ancient texts with filter cloth, and utilize pressing machine to extrude the filter cloth that fragrant plant mentioned in ancient texts is housed, collect the polysaccharide soln that plastic squeeze leaches; The polysaccharide soln of collection carried out cool, dry, to be ground into particle pack for subsequent use, be placed on shady and cool dry place and preserve, obtain fragrant plant mentioned in ancient texts Crude polysaccharides;
Step 4, ferment treatment: by the Crude polysaccharides dissolved water of cooling, make the polysaccharide soln that concentration is 5g/L, add agarase process; Enzyme concentration is 15% (v/v), and enzyme is lived as 300U/mL; Enzymolysis 20-1280min, to prepare oligosaccharide solution;
Step 5, drying: oligosaccharide solution carries out lyophilize, or vacuum-drying becomes pressed powder, obtained agaropectin oligose.
2. the fragrant plant mentioned in ancient texts dish agaropectin oligose preparation method of a kind of short lactobacillus bulgaricus growth as claimed in claim 1, it is characterized in that: in described step 4, enzymolysis time is 20min, and the obtained polymerization degree is the agaropectin oligose of 8.
3. the fragrant plant mentioned in ancient texts dish agaropectin oligose preparation method of a kind of short lactobacillus bulgaricus growth as claimed in claim 1, it is characterized in that: in described step 4, enzymolysis time is 80min, and the obtained polymerization degree is the agaropectin oligose of 5.
4. the fragrant plant mentioned in ancient texts dish agaropectin oligose preparation method of a kind of short lactobacillus bulgaricus growth as claimed in claim 1, it is characterized in that: in described step 4, enzymolysis time is 1280min, and the obtained polymerization degree is the agaropectin oligose of 3.
5. urge the application of the fragrant plant mentioned in ancient texts dish agaropectin oligose of lactobacillus bulgaricus growth as claimed in claim 1 for one kind, it is characterized in that: for low temperature storage lactobacillus bulgaricus: by lactobacillus bulgaricus by inoculum size 2%, being added into containing polymerization degree 3-8 and concentration is in the substratum of the agaropectin oligose of 1-10g/L, be placed in 42 DEG C, under oxygen free condition, quiescent culture 2-3 days, and measure viable count at set intervals.
6. urge the application of the fragrant plant mentioned in ancient texts dish agaropectin oligose of lactobacillus bulgaricus growth as claimed in claim 1 for one kind, it is characterized in that: for the preparation of the solid-state microbial inoculum of lactobacillus bulgaricus: fermentation lactobacillus bulgaricus is to a certain degree added agaropectin oligose and prepares solid-state microbial inoculum by spraying dry, and measure viable count.
7. the application of the fragrant plant mentioned in ancient texts dish agaropectin oligose of a kind of short lactobacillus bulgaricus growth as claimed in claim 6, is characterized in that: the spray parameters that described spraying dry prepares solid-state microbial inoculum is maltodextrin addition 25%, inlet temperature 130 DEG C, intake velocity 4m
3/ min, feeding rate 700mL/h, air pressure 0.3MPa.
8. urge the application of the fragrant plant mentioned in ancient texts dish agaropectin oligose of lactobacillus bulgaricus growth as claimed in claim 1 for one kind, it is characterized in that: for the effect of simulating gastrointestinal digestion to lactobacillus bulgaricus: fermentation lactobacillus bulgaricus is to a certain degree added agaropectin oligose and is positioned in in-vitro simulated human body intestinal juice or simulation human gastric juice, measure the tolerance of lactobacillus bulgaricus.
9. a kind of as claimed in claim 8 application of fragrant plant mentioned in ancient texts dish agaropectin oligose of short lactobacillus bulgaricus growth, is characterized in that: described in-vitro simulated human body intestinal juice condition is: KH
2pO
40.5%, trypsinase 0.3%, regulates pH to be 8.0,0.22 μm of filtering with microporous membrane sterilizing with NaOH.
10. the application of the fragrant plant mentioned in ancient texts dish agaropectin oligose of a kind of short lactobacillus bulgaricus growth as claimed in claim 8, it is characterized in that: described in-vitro simulated human gastric juice condition is: NaCl0.3%, KCl0.2%, stomach en-0.5%, pH is regulated to be 2.0,0.22 μm of filtering with microporous membrane sterilizing with HCl.
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| CN109820038A (en) * | 2019-04-03 | 2019-05-31 | 福州大学 | A kind of agar Yoghourt and preparation method thereof |
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