CN105105146B - A kind of activity retention method of probiotics and its application in Solid lipid food - Google Patents
A kind of activity retention method of probiotics and its application in Solid lipid food Download PDFInfo
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- CN105105146B CN105105146B CN201510605065.3A CN201510605065A CN105105146B CN 105105146 B CN105105146 B CN 105105146B CN 201510605065 A CN201510605065 A CN 201510605065A CN 105105146 B CN105105146 B CN 105105146B
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23G—COCOA; COCOA PRODUCTS, e.g. CHOCOLATE; SUBSTITUTES FOR COCOA OR COCOA PRODUCTS; CONFECTIONERY; CHEWING GUM; ICE-CREAM; PREPARATION THEREOF
- A23G1/00—Cocoa; Cocoa products, e.g. chocolate; Substitutes therefor
- A23G1/30—Cocoa products, e.g. chocolate; Substitutes therefor
- A23G1/32—Cocoa products, e.g. chocolate; Substitutes therefor characterised by the composition containing organic or inorganic compounds
- A23G1/42—Cocoa products, e.g. chocolate; Substitutes therefor characterised by the composition containing organic or inorganic compounds containing microorganisms or enzymes; containing paramedical or dietetical agents, e.g. vitamins
- A23G1/423—Cocoa products, e.g. chocolate; Substitutes therefor characterised by the composition containing organic or inorganic compounds containing microorganisms or enzymes; containing paramedical or dietetical agents, e.g. vitamins containing microorganisms, enzymes
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23G—COCOA; COCOA PRODUCTS, e.g. CHOCOLATE; SUBSTITUTES FOR COCOA OR COCOA PRODUCTS; CONFECTIONERY; CHEWING GUM; ICE-CREAM; PREPARATION THEREOF
- A23G1/00—Cocoa; Cocoa products, e.g. chocolate; Substitutes therefor
- A23G1/30—Cocoa products, e.g. chocolate; Substitutes therefor
- A23G1/32—Cocoa products, e.g. chocolate; Substitutes therefor characterised by the composition containing organic or inorganic compounds
- A23G1/44—Cocoa products, e.g. chocolate; Substitutes therefor characterised by the composition containing organic or inorganic compounds containing peptides or proteins
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Abstract
The invention discloses a kind of activity retention method of probiotics and its application in Solid lipid food, belong to food processing technology field.The present invention carries out dehydration pretreatment by osmotic balance principle using high concentration glycerine to active probiotic, to reach the purpose for reducing Residual water contained by probiotics inside.Gained activity bacterium mud is added in chocolate tempering process process, and it can be made to form good crystal structure with chocolate lipid, effectively extends vigor of the probiotics in chocolate, so as to obtain a kind of prebiotic chocolate.Activity of probiotic in the chocolate that the present invention is prepared keeps effect to be processed with lyophilized bacterium powder, and obtained product is close, and inventive process avoids high cost caused by freeze-dried powder is prepared, energy consumption is low, and equipment investment is small, can greatly save processing cost.
Description
Technical field
The present invention relates to a kind of activity retention method of probiotics and its application in Solid lipid food, belong to food
Processing technique field.
Background technology
Probiotics is a kind of active microorganism beneficial to host, can be colonized in human body intestinal canal, in reproductive system, and produce
Definite health efficacy, and then improve host's microecological balance, play the active beneficial microorganism general name of beneficial effect.In recent years
Come, with increasingly rise of the people to health attention rate, extensive pass of the probiotics by numerous scientific researchers both at home and abroad
Note, its have include improving digestive system function, enhancing is immune, reduces blood fat and blood pressure, suppresses harmful microorganism, recovery liver function
Energy, even many beneficial functions such as anti-malignant tumor.However, the performance of probiotics beneficial function need to be established in human body to a certain amount of
The intake of active somatic cell number, and active bacteria can be completed to colonize in the gastrointestinal tract.Therefore, for probiotics in food processing process
And its in storage growth activity holding, turn into the great difficult problem that food industry or even sphere of learning are faced.
The holding of bacterial activity is the physicochemical property according to microorganism, is artificially created with means such as physics, biologies suitable
Suitable condition：The water content of matrix is reduced, nutritional ingredient in culture medium is reduced, reduces partial pressure of oxygen or using low temperature, is suppressed
The respiration of microorganism, make its metabolism in it is least active, growth and breeding is suppressed or the state in geo-stationary.
The essential condition of microbial metabolism can be effectively reduced by low temperature, drying, anoxic three, microorganism is temporarily in
The state that the state of semidormancy or a complete rest are slept, so that strain is original by being maintained to after prolonged preserve
Biological characteristics and good hereditary capacity.
Ministry of Health of China was issued and defended (2001) No. 84 prebiotic mushroom health foods evaluation regulations of method prison hair the in March, 2001
Ten explicitly point out：Do not advocate and produce prebiotic mushroom health food live products in liquid form.This is due to liquid form production
Product water activity is higher, and dissolved oxygen amount is of a relatively high, and for most of microbe, its optimum moisture activity grown is
＞ 0.99.Quickly growth, metabolism and breeding, ultimately result in the quick death of strain.Therefore, the spraying of producer's generally use is dry
The drying means such as dry, freeze-drying, with reference to means such as embedding techniques, vacuum packaging and cryopreservations, to keep strain good
Growth activity.On the one hand, spray drying and Freeze Drying Technique can effectively reduce residual moisture content in strain；On the other hand,
High/low temperature and drying and dehydrating can trigger cellular damage.Embedding techniques can be combined by albumen/polysaccharide or the mode such as outer layer covers,
The external environment infringement suffered by probiotics is effectively reduced, can effectively completely cut off strain with reference to vacuum packaging and the direct of oxygen connects
Touch, effectively reduce its growth activity.However, the processing temperature of spray drying is higher, probiotics bacterium powder is made and survival be present
The more low drawback of rate；And use probiotics bacterium powder made from freeze-drying although to possess higher quality, but due to its costliness
Processing cost, seriously hinder the Popularization And Development of probiotics.Further, since dry processing technology is limited, it is on the one hand commercially available prebiotic
Becteriums product is more to be occurred in the form of dry powder state, tablet and capsule etc., and it is more single product form to be present, and generally has outside medicine
Type, consumer is easily caused to deposit the drawbacks such as mistaken ideas on concept.On the other hand, research shows existing bacterium powder dry technology to bacterium
The holding effect of vitality of subject is poor, and for usual cryopreservation after 6 months, bacterium powder vigor loss late is larger, has a strong impact on the product of product
Matter and prebiotic function.
In addition, the performance of probiotics functional activity not only needs the intake of a certain amount of active thalline, while also need to strain
Realize through digestions such as stomach and intestine and finally the long-term field planting in small intestine.Therefore, it is possible to resist stronger sour environment
It is the prerequisite that probiotics can survive and play a role in enteron aisle with possessing good resistance to bile salt ability.However, hydrochloric acid in gastric juice
Be respectively provided with stronger biocidal property with bile salt, in vitro digestion result of the test is shown, probiotics through acid and bile salt tolerance experimental test,
Thalline survival rate is generally only 40% or so, even lower.At present, it is main to use embedding techniques to improve the vigor of probiotics guarantor
Holding effect fruit, but this method is limited to embed coating material and its application vector, makes complexity, and effect is not notable.Therefore, develop
Solid-state probiotic products turn into new trend.
The content of the invention
The method of probiotic active is kept present invention firstly provides a kind of, and the activity benefit being prepared according to the method
Raw bacterium bacterium mud.
Methods described, it is by glycerite and probiotics bacterium mud (5- in mass ratio that mass fraction is 70%~90%
20)：1 mixing, after pressing to be infiltrated is weighed, active probiotic bacterium mud is collected by centrifugation.
The probiotics includes lactobacillus fermenti, lactobacillus cellobiosas, lactobacillus curvatus, lactobacillus lactis, plumule breast
The bright beading of bacillus, lactobacillus reuteri, lactobacillus acidophilus, Lactobacillus brevis, lactobacillus bulgaricus, Lactobacillus casei, mesenterium
Coccus, pediococcus cerevisiae, Pediococcus pentosaceus, lactic acid bacteria tablet coccus, streptococcus lactis, streptococcus lactis, streptococcus thermophilus, youth are double
Discrimination bacillus, animal bifidobacteria, bifidobacterium infantis, bifidobacterium longum, thermophilic Bifidobacterium etc..
In one embodiment of the invention, it is by 1~2 parts by weight concentration 2~8 × 1010Cfu/g activity bacterium mud with
The glycerine water solution that 10~20 parts by weight mass fractions are 70%~90% is mixed, and 18~24h is stood under low temperature, is then centrifuged for
Dehydration, it is 35~40% to make gained active probiotic bacterium mud water content.
In one embodiment of the invention, also containing 1~3% peptide matters in the glycerine water solution.
In one embodiment of the invention, methods described comprises the following steps：
A, activation probiotics bacterial liquid is prepared；
The probiotics bacterial liquid can be made by mixing by one or more probiotics and be formed.
Described the step of preparing activation probiotics bacterial liquid, including protected from glycerine in tube and draw probiotics bacterial liquid, it is inoculated into
In 800~1200mL MRS broth bouillons, 16~18h of activation culture is then carried out under 36~38 DEG C of temperature, anaerobic condition,
The above-mentioned activation act of repetition 2~4 times, obtain activating probiotics bacterial liquid.
Described MRS broth bouillon proportion of composing is as follows：500mL distilled water, 5g peptones, 5g beef extracts, 2.5g ferment
Female cream, 2.5g sodium acetates, 10g glucose, 0.05g MgSO4.7H20、0.025gMnSO4.H20th, 1g dibasic ammonium citrates, 1g
K2HPO4With 0.5ml tweens, pH 6.2~6.4；
B, the activation probiotics bacterial liquid for obtaining step A high speed centrifugation, separation, separation under conditions of 6000~8000g
Obtained thalline is washed 2~4 times with sterile phosphate buffer, obtains concentration 2~8 × 1010Cfu/g activity bacterium mud.
The high speed centrifugation is that 10~15min is centrifuged using high-speed centrifuge.
The proportion of composing of the phosphate buffer is as follows：0.2g KCl、0.27g KH2PO4、8.5gNaCl、2.85g
Na2HPO4·12H2O and 1000mL distilled water, pH to 7.2.
Described cfu/g meaning refers to the bacterial community sum contained in every gram of sample, the survey of its bacterial community sum
Surely conventional method well known to those skilled in the art of the present technique is used.
C, 70~90 parts by weight food grade glycerins are weighed, are added in 10~30 parts by weight of deionized water, vibration mixes, and is made
Glycerine mixed solution；
Gained activity bacterium mud in the step B is added in above-mentioned glycerine mixed solution (as above), is placed in 4 DEG C of insulating boxs
18~24h of inner equilibrium, ready to balance finish, take out, by the glycerine mixed solution containing activity bacterium mud 6000~8000g condition
Lower high speed centrifugation, separation, obtain the active probiotic bacterium mud with low Residual water.
The active probiotic bacterium mud water content of the low Residual water is 35~40%.
The Solid lipid system, main origin come from the lipid composition of animal or plant, lipid content >=70%, fusing point
>=30 DEG C, under the conditions of 35 DEG C~45 DEG C, there are good rheological properties, when being stored under the conditions of 4 DEG C~15 DEG C, be usually
Solid-state.
The present invention also provides application of the active probiotic bacterium mud in prebiotic Solid lipid food is prepared, such as chalk
Power, shortening, cheese etc., it is the later stage in Solid lipid Product processing, adds active probiotic bacterium mud and stir and evenly mix.
In order to be more convenient for understanding application of the method for the invention in Solid lipid food, the present invention also provides the work
Application process of the property probiotics bacterium mud in chocolate is prepared.
The application process, it is during being processed to different type chocolate product, by the live probiotic
Bacterium bacterium mud was added and stirred and evenly mixed in the production processing later stage.To avoid active probiotic from causing vigor because of production process
Decline or forfeiture, active probiotic is further protected in follow-up storage.
In one embodiment of the invention, the temperature in the production processing later stage should be at 35~45 DEG C, production
Product good fluidity or in viscous liquid.
In one embodiment of the invention, the application process comprises the following steps：
(1) 70%~75% cocoa liquor is weighed after high temperature melting, adds 35%~38% white sugar powder, and mixing is equal
It is even, chocolate homogenate is made, it is standby；
(2) above-mentioned chocolate is homogenized, be placed under 45~60 DEG C of temperature environment, refine 36~72h；
(3) the chocolate homogenate that refining is completed, is cooled to 40~45 DEG C, by the active probiotic bacterium mud added to ingeniously
In gram force homogenate, and complete the temperature adjustment flow of chocolate three phases.The first stage of temperature adjustment, material are cooled to 29 from 40 DEG C
℃；Second stage, 27 DEG C are cooled to from 29 DEG C；Phase III, go up from 27 DEG C to 29~30 DEG C
(4) moulding by casting：25~30min of cooling under 8~10 DEG C of environment is placed in, is stripped, packaging.
Present invention tool has the advantage that：
1. preparation method is easy, after long-time is stored, activity of probiotic keeps excellent：
The present invention using high concentration glycerine carries out dehydration pretreatment by osmotic balance principle to active probiotic, with up to
To the purpose for reducing Residual water contained by probiotics inside.After high concentration glycerine pre-processes, ICW glues somatic cells
Denseness is obviously improved, and water activity declines, and phage surface can be wrapped to form good " embedding " drop by glycerine, is formed good
Air barrier, effectively avoid thalline because of oxidative death.Activity bacterium mud after processing is applied in Solid lipid food, and
Added during Solid lipid food processing later stage tempering process, it can be made to form good crystal structure with lipid, effectively
Extend vigor of the probiotics in Solid lipid, and vigor keeps effect to be better than commercially available lyophilized bacterium powder class probiotic products, simultaneously
Avoid and prepare great number cost caused by freeze-dried powder.In addition, glycerine contributes to lipid and thalline drop as a kind of emulsifying agent
Uniform mixing.
2. in vitro digestion test result is more excellent, contribute to the field planting of probiotics enteron aisle and the performance of functional characteristic
Product is made using the method for the invention, tests and finds through in vitro digestion, it is produced with existing prebiotic mushroom
Product process technology (drying bacterium powder, embedding techniques etc.) is compared, and active thalline survival rate is obviously improved.This is mainly due to the present invention
In the Solid lipid system, probio thalline is more to be existed in the form of " oily bag bacterium ", and lipid layer is thicker, and acid and bile salt tolerance ability
By force, therefore, still there is higher survival rate after the enteral road digestion process of active thalline, contributes to field planting and the work(of probiotics enteron aisle
The performance of energy characteristic.
3. materials safety is reliable, low processing cost, equipment investment is small：
Using glycerine as osmotic pressure water balance raw material, it makes the present invention in China's GB2760-2014 food additives
In the food additives list that can need to use in right amount by producing in varieties of food items with being put into standard, therefore, do not have
Food safety hazards.Bacterium mud preprocess method is easy, and energy consumption is low, and equipment investment is small, can greatly save processing cost.
In summary, the present invention can effectively keep probiotic active, reduce the processing and fabricating cost of prebiotic becteriums product, side
Method is easy, and after long-time is stored, contained probiotics still has good growth activity, nutrient health in product.
Brief description of the drawings
The holding of Fig. 1 probiotics chocolate thalline vigor with storage time change
Embodiment
In order to preferably illustrate the advantage of explanation the method for the invention, the present invention is also to active probiotic and containing active
The chocolate of probiotics has carried out related property test, specific as follows：
According to GB/T 5497-1985 method of testings, probiotics bacterium mud reclaimed water point content is determined and compared；
According to SN/T 0168-2015 method of testings, contained probiotics in the probiotics chocolate through long-time storage is lived
Bacterium sum, which is determined, to be compared；
Activity of probiotic keeps storage experiment：Obtained probiotics chocolate is placed under 4 DEG C of environment and stored 6 months, and it is fixed
Phase carries out test and comparison to contained prebiotic bacterial content in product.
In vitro digestion is tested：The testing sample solid of appropriate quality is taken, adds simulated gastric fluid or the people of certain volume
Work intestinal juice, while control group is set, isometric PBS is added in control group, 2h is cultivated in 37 DEG C of water-baths, is run every 30min
Shake up 1 time, use gradient dilution method to carry out plate count after cultivating 2h.
Probiotics chocolate is made without the probiotic active bacterium mud that glycerine pre-processes in embodiment 1
A, protected from glycerine in tube and draw 200 μ L probiotics bacterial liquids, be inoculated into 1000mL MRS broth bouillons, then
Activation culture 17h is carried out under 37 DEG C of temperature, anaerobic condition, the above-mentioned activation act of repetition 2 times, obtains a kind of activation probiotics bacterial
Liquid；
The probiotics includes lactobacillus fermenti, lactobacillus cellobiosas, lactobacillus curvatus, lactobacillus lactis, plumule breast
The bright beading of bacillus, lactobacillus reuteri, lactobacillus acidophilus, Lactobacillus brevis, lactobacillus bulgaricus, Lactobacillus casei, mesenterium
Coccus, pediococcus cerevisiae, Pediococcus pentosaceus, lactic acid bacteria tablet coccus, streptococcus lactis, streptococcus lactis, streptococcus thermophilus, youth are double
Discrimination bacillus, animal bifidobacteria, bifidobacterium infantis, bifidobacterium longum, thermophilic Bifidobacterium.
The probiotics bacterial liquid can be made by mixing by above-mentioned one or more probiotics and be formed.
Described MRS broth bouillons composition is as follows：500mL distilled water, 5g peptones, 5g beef extracts, 2.5g yeast extracts,
2.5g sodium acetates, 10g glucose, 0.05g MgSO4·7H20、0.025gMnSO4·H20th, 1g dibasic ammonium citrates, 1g K2HPO4
With 0.5ml tweens, pH 6.2；
B, the activation bacterium solution for obtaining step A high speed centrifugation, separation, isolated thalline under conditions of 6500g is used
Sterile phosphate buffer washs 3 times, then obtains concentration 7.3 × 1010Cfu/g activity bacterium muds.
In the step B, 10min is centrifuged using high-speed centrifuge in described activation bacterium solution.
The composition of the phosphate buffer is as follows：0.2g KCl、0.27g KH2PO4、8.5gNaCl、2.85g
Na2HPO4·12H2O and 1000mL distilled water, pH to 7.2.
Described cfu/g meaning refers to the bacterial community sum contained in every gram of sample, and its bacterial community sum is adopted
Conventional method determines known to technical staff in the art.
The active probiotic bacterium mud water content is 80.92%.
C, chocolate is prepared
(1) 70% cocoa liquor is weighed after high temperature melting, adds 36% white sugar powder, is well mixed, and chocolate is made
Homogenate, it is standby；
(2) above-mentioned chocolate is homogenized, be placed under 50 DEG C of temperature environment, refine 40h；
(3) the chocolate homogenate that refining is completed, is cooled to 40 DEG C, by live probiotic in the method for the invention step B
During bacterium bacterium mud is homogenized added to chocolate, and complete the temperature adjustment flow of chocolate three phases.The first stage of temperature adjustment, material from
40 DEG C are cooled to 29 DEG C；Second stage, 27 DEG C are cooled to from 29 DEG C；Phase III, go up from 27 DEG C to 29 DEG C；
(4) moulding by casting, it is placed under 8 DEG C of environment and cools down 25min, is stripped, packaging.
Embodiment 2 freezes bacterium powder and probiotics chocolate is made
A, protected from glycerine in tube and draw 200 μ L probiotics bacterial liquids, be inoculated into 1000mL MRS broth bouillons, then
Activation culture 17h is carried out under 37 DEG C of temperature, anaerobic condition, the above-mentioned activation act of repetition 2 times, obtains a kind of activation probiotics bacterial
Liquid；
The probiotics includes lactobacillus fermenti, lactobacillus cellobiosas, lactobacillus curvatus, lactobacillus lactis, plumule breast
The bright beading of bacillus, lactobacillus reuteri, lactobacillus acidophilus, Lactobacillus brevis, lactobacillus bulgaricus, Lactobacillus casei, mesenterium
Coccus, pediococcus cerevisiae, Pediococcus pentosaceus, lactic acid bacteria tablet coccus, streptococcus lactis, streptococcus lactis, streptococcus thermophilus, youth are double
Discrimination bacillus, animal bifidobacteria, bifidobacterium infantis, bifidobacterium longum, thermophilic Bifidobacterium.
The probiotics bacterial liquid can be made by mixing by above-mentioned one or more probiotics and be formed.
Described MRS broth bouillons composition is as follows：500mL distilled water, 5g peptones, 5g beef extracts, 2.5g yeast extracts,
2.5g sodium acetates, 10g glucose, 0.05g MgSO4·7H20、0.025gMnSO4·H20th, 1g dibasic ammonium citrates, 1g K2HPO4
With 0.5ml tweens, pH 6.2；
B, the activation bacterium solution for obtaining step A high speed centrifugation, separation, isolated thalline under conditions of 6500g is used
Sterile phosphate buffer washs 3 times, then obtains concentration 7.3 × 1010Cfu/g activity bacterium muds.
In the step B, 10min is centrifuged using high-speed centrifuge in described activation bacterium solution.
The composition of the phosphate buffer is as follows：0.2g KCl、0.27g KH2PO4、8.5gNaCl、2.85g
Na2HPO4·12H2O and 1000mL distilled water, pH to 7.2.
Described cfu/g meaning refers to the bacterial community sum contained in every gram of sample, and its bacterial community sum is adopted
Conventional method determines known to technical staff in the art.
C, probiotics freezes the making of bacterium powder
By activity bacterium mud described in step B, 30 parts by weight degreasing milk solutions are added, are well mixed, then it is freeze-dried, obtain
Bacterium powder is freezed to high-activity probiotics, it is standby, while for storing the measure of experiment viable count.
It is 8.9 × 10 that the probiotics, which freezes bacterium powder total plate count,9cfu/g；
It is 8.21% that the probiotics, which freezes bacterium powder moisture,.
D, chocolate is prepared
(1) 70% cocoa liquor is weighed after high temperature melting, adds 36% white sugar powder, is well mixed, and chocolate is made
Homogenate, it is standby；
(2) above-mentioned chocolate is homogenized, be placed under 50 DEG C of temperature environment, refine 40h；
(3) the chocolate homogenate that refining is completed, is cooled to 40 DEG C, by live probiotic in the method for the invention step C
Bacterium freezes bacterium powder and is added in chocolate homogenate, and completes the temperature adjustment flow of chocolate three phases.The first stage of temperature adjustment, thing
Material is cooled to 29 DEG C from 40 DEG C；Second stage, 27 DEG C are cooled to from 29 DEG C；Phase III, go up from 27 DEG C to 29 DEG C；
(4) moulding by casting, it is placed under 8 DEG C of environment and cools down 25min, is stripped, packaging.
Active probiotic made from the inventive method of embodiment 3 is used to prepare probiotics chocolate
A, protected from glycerine in tube and draw 200 μ L probiotics bacterial liquids, be inoculated into 1000mL MRS broth bouillons, then
Activation culture 17h is carried out under 37 DEG C of temperature, anaerobic condition, the above-mentioned activation act of repetition 2 times, obtains a kind of activation probiotics bacterial
Liquid；
The probiotics includes lactobacillus fermenti, lactobacillus cellobiosas, lactobacillus curvatus, lactobacillus lactis, plumule breast
The bright beading of bacillus, lactobacillus reuteri, lactobacillus acidophilus, Lactobacillus brevis, lactobacillus bulgaricus, Lactobacillus casei, mesenterium
Coccus, pediococcus cerevisiae, Pediococcus pentosaceus, lactic acid bacteria tablet coccus, streptococcus lactis, streptococcus lactis, streptococcus thermophilus, youth are double
Discrimination bacillus, animal bifidobacteria, bifidobacterium infantis, bifidobacterium longum, thermophilic Bifidobacterium.
The probiotics bacterial liquid can be made by mixing by above-mentioned one or more probiotics and be formed.
Described MRS broth bouillons composition is as follows：500mL distilled water, 5g peptones, 5g beef extracts, 2.5g yeast extracts,
2.5g sodium acetates, 10g glucose, 0.05g MgSO4·7H20、0.025gMnSO4·H20th, 1g dibasic ammonium citrates, 1g K2HPO4
With 0.5ml tweens, pH 6.2；
B, the activation bacterium solution for obtaining step A high speed centrifugation, separation, isolated thalline under conditions of 6500g is used
Sterile phosphate buffer washs 3 times, then obtains concentration 7.3 × 1010Cfu/g activity bacterium muds.
In the step B, 10min is centrifuged using high-speed centrifuge in described activation bacterium solution.
The composition of the phosphate buffer is as follows：0.2g KCl、0.27g KH2PO4、8.5gNaCl、2.85g
Na2HPO4·12H2O and 1000mL distilled water, pH to 7.2.
Described cfu/g meaning refers to the bacterial community sum contained in every gram of sample, and its bacterial community sum is adopted
Conventional method determines known to technical staff in the art.
C, 90 parts by weight food grade glycerins are weighed, are added in 10 parts by weight of deionized water, vibration mixes, and glycerine mixing is made
Solution；
Gained activity bacterium mud in the step B is added in above-mentioned glycerine mixed solution, is placed in 4 DEG C of insulating box inner equilibriums
20h, ready to balance finish, and take out, high speed centrifugation, separation under conditions of 6500g by the glycerine mixed solution containing activity bacterium mud,
The active probiotic bacterium mud with low Residual water is obtained, for the processed and applied in chocolate；
The active probiotic bacterium mud water content of the low Residual water is 35.67%.
D, chocolate is prepared
(1) 70% cocoa liquor is weighed after high temperature melting, adds 36% white sugar powder, is well mixed, and chocolate is made
Homogenate, it is standby；
(2) above-mentioned chocolate is homogenized, be placed under 50 DEG C of temperature environment, refine 40h；
(3) the chocolate homogenate that refining is completed, is cooled to 40 DEG C, by low remaining water in the method for the invention step C
During the active probiotic bacterium mud divided is homogenized added to chocolate, and complete the temperature adjustment flow of chocolate three phases.The of temperature adjustment
One stage, material are cooled to 29 DEG C from 40 DEG C；Second stage, 27 DEG C are cooled to from 29 DEG C；Phase III, go up from 27 DEG C to 29
℃；
(4) moulding by casting, it is placed under 8 DEG C of environment and cools down 25min, is stripped, packaging.
Active probiotic made from the inventive method of embodiment 4 is used to prepare probiotics chocolate
A, protected from glycerine in tube and draw 200 μ L probiotics bacterial liquids, be inoculated into 1200mL MRS broth bouillons, then
Activation culture 18h is carried out under 38 DEG C of temperature, anaerobic condition, the above-mentioned activation act of repetition 2 times, obtains a kind of activation probiotics bacterial
Liquid；
The probiotics includes lactobacillus fermenti, lactobacillus cellobiosas, lactobacillus curvatus, lactobacillus lactis, plumule breast
The bright beading of bacillus, lactobacillus reuteri, lactobacillus acidophilus, Lactobacillus brevis, lactobacillus bulgaricus, Lactobacillus casei, mesenterium
Coccus, pediococcus cerevisiae, Pediococcus pentosaceus, lactic acid bacteria tablet coccus, streptococcus lactis, streptococcus lactis, streptococcus thermophilus, youth are double
Discrimination bacillus, animal bifidobacteria, bifidobacterium infantis, bifidobacterium longum, thermophilic Bifidobacterium.
The probiotics bacterial liquid can be made by mixing by above-mentioned one or more probiotics and be formed.
Described MRS broth bouillons composition is as follows：500mL distilled water, 5g peptones, 5g beef extracts, 2.5g yeast extracts,
2.5g sodium acetates, 10g glucose, 0.05g MgSO4·7H20、0.025gMnSO4·H20th, 1g dibasic ammonium citrates, 1g K2HPO4
With 0.5ml tweens, pH 6.3；
B, the activation bacterium solution for obtaining step A high speed centrifugation, separation, isolated thalline under conditions of 8000g is used
Sterile phosphate buffer washs 4 times, then obtains concentration 6.3 × 1010Cfu/g activity bacterium muds.
In the step B, 15min is centrifuged using high-speed centrifuge in described activation bacterium solution.
The composition of the phosphate buffer is as follows：0.2g KCl、0.27g KH2PO4、8.5gNaCl、2.85g
Na2HPO4·12H2O and 1000mL distilled water, pH to 7.2.
Described cfu/g meaning refers to the bacterial community sum contained in every gram of sample, and its bacterial community sum is adopted
Conventional method determines known to technical staff in the art.
C, 75 parts by weight food grade glycerins and 1.6 parts by weight peptide matters are weighed, adds in 25 parts by weight of deionized water, shakes
Mixing is swung, glycerine mixed solution is made；
Gained activity bacterium mud in the step B is added in above-mentioned glycerine mixed solution, is placed in 4 DEG C of insulating box inner equilibriums
24h, ready to balance finish, and take out, high speed centrifugation, separation under conditions of 8000g by the glycerine mixed solution containing activity bacterium mud,
The active probiotic bacterium mud with low Residual water is obtained, for the processed and applied in chocolate；
The active probiotic bacterium mud water content of the low Residual water is 36.06%.
D, chocolate is prepared
(1) 75% cocoa liquor is weighed after high temperature melting, adds 38% white sugar powder, is well mixed, and chocolate is made
Homogenate, it is standby；
(2) above-mentioned chocolate is homogenized, be placed under 45 DEG C of temperature environment, refine 60h；
(3) the chocolate homogenate that refining is completed, is cooled to 45 DEG C, by low remaining water in the method for the invention step C
During the active probiotic bacterium mud divided is homogenized added to chocolate, and complete the temperature adjustment flow of chocolate three phases.The of temperature adjustment
One stage, material are cooled to 29 DEG C from 40 DEG C；Second stage, 27 DEG C are cooled to from 29 DEG C；Phase III, go up from 27 DEG C to 30
℃；
(4) moulding by casting, it is placed under 10 DEG C of environment and cools down 30min, is stripped, packaging.
The comparison of probiotics moisture under the conditions of the different disposal of table 1.
Influence of the glycerine of the table 2. balance pretreatment to growth of probiotics activity
External tolerance experimental results of the table 3 in simulated gastric fluid and simulated intestinal fluid
It will be seen from figure 1 that using chocolate made from probiotics after long-time storage is placed, using fresh bacterium mud system
For total amount of probiotics with the extension of storage time, decline is more apparent, and uses the inventive method and lyophilized bacterium powder in the chocolate obtained
Obtained chocolate, contained total amount of probiotics does not decline significantly with the extension of storage time in it, and bacterium after 30 days
Fall sum to tend towards stability.In addition, embodiment 3,4 gained chocolate flavourings are not influenceed by glycerine.
Although the present invention is disclosed as above with preferred embodiment, it is not limited to the present invention, any to be familiar with this skill
The people of art, without departing from the spirit and scope of the present invention, it can all do various change and modification, therefore the protection model of the present invention
Enclose being defined of being defined by claims.
Claims (9)
- A kind of 1. method for preparing prebiotic Solid lipid product, it is characterised in that be in the later stage of Solid lipid Product processing, add Enter active probiotic bacterium mud and stir and evenly mix；The active probiotic bacterium mud be by by mass fraction be 70%~90% it is sweet Oil solution is 2~8 × 10 with concentration10Cfu/g probiotics bacterium mud (5-20) in mass ratio：1 mixing, after pressing to be infiltrated is weighed, Obtained bacterium mud is collected by centrifugation；The temperature in the later stage of the Solid lipid Product processing is in 40~45 DEG C, by the activity benefit During raw bacterium bacterium mud is homogenized added to chocolate, and complete the temperature adjustment flow of chocolate three phases；The temperature adjustment flow includes：Adjust The first stage of temperature, material are cooled to 29 DEG C from 40 DEG C, second stage, 27 DEG C are cooled to from 29 DEG C, the phase III, is returned from 27 DEG C Rise to 29~30 DEG C；(4) moulding by casting：25~30min of cooling under 8~10 DEG C of environment is placed in, is stripped, packaging, is obtained prebiotic skilful Gram force.
- 2. according to the method for claim 1, it is characterised in that the Solid lipid product is chocolate, shortening or milk Junket.
- 3. according to the method for claim 1, it is characterised in that the probiotics includes lactobacillus fermenti, cellobiose breast Bacillus, lactobacillus curvatus, lactobacillus lactis, lactobacillus plantarum, lactobacillus reuteri, lactobacillus acidophilus, Lactobacillus brevis, Bao Jiali The bright beading coccus of sub- lactobacillus, Lactobacillus casei, mesenterium, pediococcus cerevisiae, Pediococcus pentosaceus, lactic acid bacteria tablet coccus, lactic acid Streptococcus, streptococcus lactis, streptococcus thermophilus, bifidobacterium adolescentis, animal bifidobacteria, bifidobacterium infantis, bifidobacterium longum, Thermophilic Bifidobacterium.
- 4. according to any described methods of claim 1-3, it is characterised in that the active probiotic bacterium mud is by 1~2 weight Part concentration 2~8 × 1010Cfu/g activity bacterium mud and the glycerine water solution that 10~20 parts by weight mass fractions are 70%~90% Mixing, 18~24h is stood under low temperature, be then centrifuged for dehydration and collect bacterium mud；The active probiotic bacterium mud water content be 35~ 40%.
- 5. according to the method for claim 2, it is characterised in that comprise the following steps：(1) 70%~75% cocoa liquor is weighed after high temperature melting, adds 35%~38% white sugar powder, is well mixed, system Chocolate homogenate is obtained, it is standby；(2) above-mentioned chocolate is homogenized, be placed under 45~60 DEG C of temperature environment, refine 36~72h；(3) the chocolate homogenate that refining is completed, is cooled to 40~45 DEG C, the active probiotic bacterium mud is added into chocolate In homogenate, and complete the temperature adjustment flow of chocolate three phases；The temperature adjustment flow includes：The first stage of temperature adjustment, material from 40 DEG C are cooled to 29 DEG C, second stage, 27 DEG C are cooled to from 29 DEG C, the phase III, is gone up from 27 DEG C to 29~30 DEG C；(4) moulding by casting：25~30min of cooling under 8~10 DEG C of environment is placed in, is stripped, packaging, obtains prebiotic chocolate.
- 6. according to the method for claim 1, it is characterised in that in the glycerite also containing as mass fraction 1~ 3% peptide matters.
- 7. the prebiotic Solid lipid product being prepared according to claim 1 or 2 or 3 or 4 or 6 methods describeds.
- 8. it is a kind of keep probiotic active method, it is characterised in that be by mass fraction be 70%~90% glycerite It is 2~8 × 10 with concentration10Cfu/g probiotics bacterium mud (5-20) in mass ratio：1 mixing, 18~24h is stood under low temperature, is treated Osmotic balance, active probiotic bacterium mud is collected by centrifugation；The active probiotic bacterium mud water content is 35~40%；It is described prebiotic Bacterium bag includes lactobacillus fermenti, lactobacillus cellobiosas, lactobacillus curvatus, lactobacillus lactis, lactobacillus plantarum, Luo Yishi breast bars The bright beading coccus of bacterium, lactobacillus acidophilus, Lactobacillus brevis, lactobacillus bulgaricus, Lactobacillus casei, mesenterium, pediococcus cerevisiae, Pediococcus pentosaceus, lactic acid bacteria tablet coccus, streptococcus lactis, streptococcus lactis, streptococcus thermophilus, bifidobacterium adolescentis, animal bifid Bacillus, bifidobacterium infantis, bifidobacterium longum, thermophilic Bifidobacterium.
- 9. the active probiotic bacterium mud that method is prepared according to claim 8.
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| CN107455538A (en) * | 2017-07-14 | 2017-12-12 | 江苏微康生物科技有限公司 | A kind of probiotics marble chocolate and preparation method thereof |
| CN108456645A (en) * | 2018-05-04 | 2018-08-28 | 北京中特养生物技术研究所有限公司 | A kind of store method of probiotics |
| CN109259109A (en) * | 2018-10-26 | 2019-01-25 | 福建农林大学 | A method of red-cooked pork flavoring bag flavor is kept based on microbial fermentation |
| CN109480038B (en) * | 2018-12-21 | 2024-10-11 | 杭州远大生物制药有限公司 | Temperature-resistant probiotic chocolate product and preparation method thereof |
| CN110122564A (en) * | 2019-04-25 | 2019-08-16 | 华南理工大学 | A kind of probiotics liposome and preparation method thereof |
| CN110711182A (en) * | 2019-10-29 | 2020-01-21 | 上海交通大学医学院附属仁济医院 | Surface-modified microorganism, and preparation method and application thereof |
| CN110903980A (en) * | 2019-11-25 | 2020-03-24 | 河南大学 | Method for preserving live lactococcus and live lactococcus preparation |
| CN110903981A (en) * | 2019-11-25 | 2020-03-24 | 河南大学 | Method for preserving live lactobacillus and live lactobacillus preparation |
| CN111187734A (en) * | 2019-12-25 | 2020-05-22 | 生合生物科技（扬州）有限公司 | Bifidobacterium longum BL986, freeze-dried powder prepared from bifidobacterium longum BL986 and application of freeze-dried powder |
| CN112553128B (en) * | 2020-12-30 | 2023-08-18 | 瞿瀚鹏 | Probiotic freeze-dried powder and preparation method and application thereof |
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