CN111518728B - Nano-phytoglycogen lactobacillus composite protective agent, preparation method and application thereof - Google Patents

Nano-phytoglycogen lactobacillus composite protective agent, preparation method and application thereof Download PDF

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CN111518728B
CN111518728B CN202010394547.XA CN202010394547A CN111518728B CN 111518728 B CN111518728 B CN 111518728B CN 202010394547 A CN202010394547 A CN 202010394547A CN 111518728 B CN111518728 B CN 111518728B
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王劲松
朱卓英
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Abstract

The invention discloses a nano-phytoglycogen lactobacillus composite protective agent, a preparation method and application thereof, and belongs to the technical field of preparation of microbial protective agents. The nano phytoglycogen lactic acid bacteria compound protective agent comprises skim milk powder, water-soluble nano phytoglycogen, Pickering emulsion of hydrophobic nano phytoglycogen and normal saline. The lactobacillus bacterial mud obtained by fermentation and centrifugation is added into the nano phytoglycogen lactobacillus composite protective agent prepared by the method, the mixture is uniformly mixed and sprayed on the feed, the survival rate of the lactobacillus in the feed can be obviously improved, and in-vitro simulated digestion experiments prove that the lactobacillus added with the protective agent has an obvious survival rate in artificial gastrointestinal fluids and a better gastrointestinal resistance effect compared with the lactobacillus not added with the protective agent, and is beneficial to the function of the lactobacillus bacterial mud in human or animals.

Description

Nano-phytoglycogen lactobacillus composite protective agent, preparation method and application thereof
Technical Field
The invention belongs to the technical field of preparation of microbial protectants, and particularly relates to a nano phytoglycogen lactobacillus composite protectant, and a preparation method and application thereof.
Background
Lactic Acid Bacteria (LAB) are a general term for a group of bacteria that can utilize fermentable carbohydrates to produce large amounts of lactic acid. Lactic acid bacteria as beneficial bacteria can improve the flavor of food, increase the nutritive value, and play a positive role in human health in many aspects, such as maintaining the balance of intestinal flora, secreting antigens, relieving lactose intolerance, enhancing the immunity of the organism, reducing the activity of serum cholesterol, inhibiting the absorption of cholesterol, preventing cancer, resisting hypertension, treating various human intestinal dysfunction and functional disorders such as acute gastroenteritis, food allergy, atopic dermatitis, rheumatoid arthritis, and the like. Furthermore, lactic acid bacteria added to animal feed can reduce the incidence of disease in animals and improve their intestinal flora and promote their growth.
At present, lactic acid bacteria are increasingly widely applied to industries such as food, medicine, livestock and poultry production and the like. However, the lactobacillus bacteria liquid has the limitations of short storage life, low stability, difficult transportation and the like, and the lactobacillus protective agent can make up the limitations, effectively protect lactobacillus cells, improve the survival rate of the lactobacillus cells and improve the application of the lactobacillus. The protective agent can reduce the damage to cells caused by dehydration in the preparation and storage processes, can provide physical support for the encapsulated and dried thalli, and can reduce the damage in the chemical aspect, so that the original characteristics of the cells can be kept as much as possible, and therefore, the selection of a proper protective agent is very important for the viability of lactic acid bacteria. However, the existing protective agent has the defects of high cost, low strain survival rate, undesirable protective effect and the like.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides a nano-phytoglycogen lactobacillus composite protective agent, a preparation method and application thereof.
In order to achieve the purpose, the invention adopts the following technical scheme:
a nanometer phytoglycogen lactobacillus composite protectant comprises skimmed milk powder, water-soluble nanometer phytoglycogen, Pickering emulsion of hydrophobic nanometer phytoglycogen and normal saline;
wherein the content of the first and second substances,
the content of the skimmed milk powder in the composite protective agent is 5-15% (w/w);
the content of the water-soluble nano phytoglycogen in the composite protective agent is 0.5-1.5% (w/w);
the addition amount of the Pickering emulsion of the hydrophobic nano phytoglycogen in the composite protective agent is 5-15% (w/w);
the balance being normal saline.
On the basis of the scheme, the water-soluble nano phytoglycogen is prepared by the following method:
weighing sweet corn seeds, adding deionized water, and soaking at 4 ℃ for 24 h; removing the water solution from the soaked corn seeds, crushing, and then mixing the corn seeds according to the proportion of 1: 3(v/v) adding deionized water, and standing for 24h at 4 ℃; taking the supernatant, adding acetic acid to adjust the pH to 4.8-5, and standing at 4 deg.C for 24 hr; centrifuging at 3000g for 10min to remove precipitate, boiling the obtained supernatant, centrifuging at 3000g for 10min again to further remove protein until no oil exists and no precipitate exists; adding absolute ethanol into the obtained supernatant according to the volume of 1:1, stirring and mixing, standing at 4 ℃ until the precipitate is complete, centrifuging 3000g, taking the precipitate, and freeze-drying to obtain the water-soluble nano phytoglycogen.
On the basis of the scheme, the preparation method of the Pickering emulsion of the hydrophobic nano phytoglycogen comprises the following steps: mixing the hydrophobic nano-phytoglycogen with deionized water until the concentration is 5% (w/w), adding medium-chain triglyceride to 50% (w/w), homogenizing at 18000rpm for 4 minutes at a high speed under the pH value of 7.0, and thus obtaining the Pickering emulsion of the hydrophobic nano-phytoglycogen.
On the basis of the scheme, the preparation method of the hydrophobic nano phytoglycogen comprises the following steps:
diluting octenyl succinic anhydride to 20% (w/w) in isopropanol, adding into 30% (w/w) water-soluble nano-phytoglycogen suspension, and diluting to 10% (w/w); adjusting the pH value to 8.5 by using a 3% NaOH solution, fully reacting for 2 hours, and adjusting the pH value to 6.5 by using 2.5M hydrochloric acid; keeping the temperature at 35 ℃ for 8 hours, centrifuging 3000g for 10 minutes after the culture is finished, taking the precipitate, washing the precipitate by distilled water and absolute ethyl alcohol in sequence, and repeating the steps for 3 times to obtain the hydrophobic nano-phytoglycogen.
The preparation method of the nano-phytoglycogen lactic acid bacteria compound protective agent comprises the following steps:
(1) mixing sterilized skimmed milk powder with 0.85% of autoclaved normal saline, and shaking and mixing to obtain the first layer of protective agent.
(2) Adding the sterilized and freeze-dried water-soluble nano phytoglycogen into the first layer of protective agent, and uniformly oscillating to form a compact second layer of protective solution;
(3) adding Pickering emulsion of hydrophobic nano-phytoglycogen into the second layer of protective solution, adjusting the pH to 7, homogenizing at 18000rpm for 4 minutes at high speed, and forming a third layer of nano-phytoglycogen lactic acid bacteria composite protective solution to obtain the nano-phytoglycogen lactic acid bacteria composite protective agent.
On the basis of the scheme, the content of the skimmed milk powder in the composite protective agent is 10 percent;
the content of the water-soluble nano phytoglycogen in the composite protective agent is 1% (w/w);
the addition amount of the Pickering emulsion of the hydrophobic nano phytoglycogen in the composite protective agent is 10% (w/w).
The application of the nano-phytoglycogen lactobacillus composite protective agent prepared by the method in preparing a lactobacillus preparation is used for improving the survival rate of lactobacillus and improving the tolerance of the lactobacillus to gastric juice and intestinal juice.
A lactobacillus preparation is prepared by adding lactobacillus mud obtained by fermentation and centrifugation into the composite protectant containing nano-phytoglycogen lactobacillus, and mixing to make lactobacillus concentration be 109cfu/mL。
The application of the lactobacillus preparation in preparing lactobacillus feed.
A fish feed containing lactobacillus is characterized in that the fish feed containing lactobacillus is obtained by spraying the lactobacillus preparation on the fish feed; wherein the spraying amount of the nano plant glycogen lactobacillus composite protective agent is 100 mu L/g feed.
The invention has the beneficial effects that:
the main component of the protective agent of the invention, namely the nano phytoglycogen is extracted from the sweet corn, the extraction rate is 17 percent, and the cost is greatly reduced; the performance of the lactobacillus protective agent improved by adding the nano particles is initiated at home and abroad; and the used protective agent components including phytoglycogen and skimmed milk powder are natural edible raw materials, so that the safety of the protective agent is guaranteed. Meanwhile, experiments prove that the composite protective agent is used for preserving the feed for 1 week, so that the survival rate of the lactobacillus in the feed is improved by more than 6 times, and the survival rate of the lactobacillus is remarkably improved. The in vitro simulated digestion experiment proves that the lactobacillus added with the protective agent has obvious survival rate in artificial gastrointestinal fluids and better gastrointestinal resistance effect compared with the lactobacillus not added with the protective agent, and is beneficial to the function of the lactobacillus in human or animals.
Drawings
FIG. 1 shows a blank (bacterial suspension without protective solution);
FIG. 2 is a single layer 10% skimmed milk powder protective solution;
FIG. 3 is a two-layer protective solution of 10% skimmed milk powder and 1% water-soluble nano phytoglycogen;
FIG. 4 is a Pickering emulsion composite protective solution of 10% skimmed milk powder, 1% phytoglycogen and 10% hydrophobic nano-phytoglycogen;
FIG. 5 shows the effect of water-soluble nano-phytoglycogen on 10% skim milk powder protectant at different concentrations, with different letters indicating significant difference (P < 0.05).
FIG. 6 is a transmission electron microscope image of phytoglycogen nanoparticles.
Fig. 7 shows the effect of Pickering emulsions of different concentrations of hydrophobic nano phytoglycogen on the colony count of lactobacillus feed, with different letters indicating significant differences in the same column (P < 0.05).
Fig. 8 shows the effect of different protective solutions on the number of colonies of the lactic acid bacteria feed, with different letters indicating significant differences (P < 0.05).
Detailed Description
Terms used in the present invention have generally meanings as commonly understood by one of ordinary skill in the art, unless otherwise specified.
The present invention will be described in further detail with reference to the following data in conjunction with specific examples. The following examples are intended to illustrate the invention and are not intended to limit the scope of the invention in any way.
Example 1
A nanometer phytoglycogen lactobacillus composite protectant comprises skimmed milk powder, water-soluble nanometer phytoglycogen, Pickering emulsion of hydrophobic nanometer phytoglycogen and normal saline;
wherein the content of the first and second substances,
the content of the skimmed milk powder in the composite protective agent is 10% (w/w);
the content of the water-soluble nano phytoglycogen in the composite protective agent is 1% (w/w);
the addition amount of the Pickering emulsion of the hydrophobic nano phytoglycogen in the composite protective agent is 10% (w/w);
the balance being normal saline.
(1) The preparation method of the water-soluble nano phytoglycogen suspension comprises the following steps:
weighing sweet corn seeds, adding deionized water, and soaking at 4 deg.C for 24 hr. Removing the aqueous solution, crushing by a pulverizer, and then performing the following steps of 1: 3(v/v) adding deionized water, and standing for 24h at 4 ℃; taking supernatant, adding acetic acid to adjust pH to 4.8-5, and standing at 4 deg.C for 24 hr; centrifuging at 3000g for 10min to remove precipitate, boiling the obtained supernatant in boiling water bath, centrifuging at 3000g for 10min again to further remove protein until no oil or precipitate exists; adding anhydrous ethanol into the obtained supernatant, stirring and mixing, mixing the supernatant and the anhydrous ethanol according to the volume of 1:1, standing at 4 ℃ until the precipitate is completely precipitated, centrifuging 3000g, and freeze-drying the precipitate (at-55 ℃ and the vacuum degree of 0.026mbar) to obtain the water-soluble nano phytoglycogen.
And dissolving the water-soluble nano phytoglycogen into deionized water to obtain 30% (w/w) of water-soluble nano phytoglycogen suspension for later use.
(2) Preparation method of Pickering emulsion of hydrophobic nano phytoglycogen
Octenyl Succinic Anhydride (OSA) is diluted to 20% (w/w) in isopropanol and added into 30% (w/w) of water-soluble nano-phytoglycogen suspension to dilute the concentration of the water-soluble nano-phytoglycogen suspension to 10% (w/w). The pH was adjusted to 8.5 with 3% NaOH solution, reacted thoroughly for 2h, and adjusted to 6.5 with 2.5M hydrochloric acid. The cells were incubated at 35 ℃ for 8 hours. And centrifuging at 3000g for 10min after the culture is finished, taking the precipitate, washing with distilled water and absolute ethyl alcohol sequentially, repeating for 3 times, collecting the hydrophobic nano-phytoglycogen, and further analyzing.
Preparing a Pickering emulsion of hydrophobic nano phytoglycogen: mixing the hydrophobic phytoglycogen nanoparticles in the step (i) with deionized water until the concentration is 5% (w/w). Then adding medium chain triglyceride to 50% (w/w), homogenizing at 18000rpm for 4 minutes at pH7.0 to obtain Pickering emulsion of hydrophobic nano-phytoglycogen.
Example 2
The preparation method of the nano-phytoglycogen lactic acid bacteria compound protective agent comprises the following steps:
(1) the water-soluble nano phytoglycogen sterilization method comprises the following steps: the solution was sterilized by filtration and filtered through a 0.22 μm water-soluble filter.
(2) The sterilization method of the skimmed milk powder comprises the following steps: sterilizing by pasteurization, heating the sample in metal bath to 68-70 deg.C for 30min, and rapidly cooling to 4-5 deg.C.
(3) Mixing sterilized skimmed milk powder with 0.85% of autoclaved normal saline, and shaking and mixing uniformly to form a first layer of protective agent;
(4) adding the sterilized and freeze-dried water-soluble nano phytoglycogen into the first layer of protective agent, and uniformly oscillating to form a compact second layer of protective solution;
(5) adding Pickering emulsion of hydrophobic nano-phytoglycogen into the second layer of protective solution, adjusting the pH to 7, homogenizing at 18000rpm for 4 minutes at high speed, and forming a third layer of nano-phytoglycogen lactic acid bacteria composite protective solution to obtain the nano-phytoglycogen lactic acid bacteria composite protective agent.
The content of the skimmed milk powder in the composite protective agent is 10 percent;
the content of the water-soluble nano phytoglycogen in the composite protective agent is 1% (w/w);
the addition amount of the Pickering emulsion of the hydrophobic nano phytoglycogen in the composite protective agent is 10% (w/w).
Example 3
A lactobacillus preparation is prepared by adding lactobacillus mud obtained by fermentation and centrifugation into the nano-phytoglycogen lactobacillus composite protective agent prepared by the method of example 2, and mixing uniformly to make the concentration of lactobacillus be 109cfu/mL. The method comprises the following specific steps:
putting 50 mu L of Lactobacillus casei K17(Lactobacillus casei K17 is preserved in China center for type culture Collection in 2018, 11 and 29 days, with the preservation number of CCTCC NO: M2018840, requesting to preserve people, Shanghai university of sea, the preservation address: China center for type culture Collection in Mega mountain, Wuhan city, Wuchang Lojiashan) into a test tube containing 5mL of sterilized MRS liquid medium, resuscitating in a 37 ℃ incubator for 18 hours to allow K17 to enter a stabilization period, respectively inoculating according to 4% of inoculation, centrifuging for 10min at 4000rpm/min and 4 ℃ after secondary passage, then discarding the supernatant, mixing bacterial sludge with PBS, putting 200 mu L into a 96-well microplate reader, measuring OD (optical density) of the mixture, and collecting the mixture600nmCentrifuging to remove PBS to obtain initial OD of about 1, adding the compound protectant of nano-phytoglycogen lactic acid bacteria prepared in example 2, mixing, and adding the final compound protectant of nano-phytoglycogen lactic acid bacteria in an amount equal to the removed PBSThe concentration of bacteria is about 109cfu/mL。
Observation of thallus by scanning electron microscope
(1) Observing the bacterium powder by a scanning electron microscope, sticking the freeze-dried bacterium powder on a sample table of the scanning electron microscope by using a double-sided adhesive tape, and carrying out gold spraying and observation under the electron microscope.
(2) Observing the microstructure of the thallus by a scanning electron microscope, rehydrating the freeze-dried powder by using 0.1mol/L sterile phosphate buffer solution with the pH value of 7.0, centrifuging for 10min at the speed of 5000 r/min, and removing the supernatant. The samples were fixed in 2.5% glutaraldehyde solution at 4 ℃ overnight and treated with 20%, 40%, 60%, 80% and 95% ethanol solution in a gradient dehydration process for 15min, while treated with 100% ethanol 2 times for 20min each time, during which the cells were collected by centrifugation at 5000 r/min for 5 min. Vacuum drying at 40 deg.C, spraying gold film, observing the morphological characteristics of thallus under scanning electron microscope, and taking photograph. FIG. 1 shows a blank (bacterial suspension without protective solution); FIG. 2 is a single layer 10% skimmed milk powder protective solution; FIG. 3 is a two-layer protective solution of 10% skimmed milk powder and 1% water-soluble nano phytoglycogen; FIG. 4 shows a Pickering emulsion composite protective solution containing 10% skimmed milk powder, 1% phytoglycogen and 10% hydrophobic nano-phytoglycogen.
Example 4
Example 3 use of the lactic acid bacteria preparation prepared in Fish feed
20g of micropterus salmoides feed (self-made by Shanghai ocean university aquatic product and Life academy animal nutrition and feed laboratories) is put into a sterilization bag and sealed, sterilized at 121 ℃ and autoclaved for 20 minutes, dried in a 65 ℃ oven for 2-3 hours (the sterilization bag is observed to be in an anhydrous state by naked eyes), weighed by an electronic balance in a super-clean workbench and subpackaged into 8 culture dishes, wherein each culture dish is about 1g, and the culture dishes are sealed for later use.
The micropterus salmoides feed formula (weight fraction): 35% of fish meal, 8% of soybean protein concentrate, 8% of soybean meal, 8% of corn protein, 18.95% of flour, 4% of wheat gluten, 4% of beer yeast, 3% of fish oil, 3% of soybean lecithin, 2% of squid liver paste, 2.5% of mineral premix, 0.5% of vitamin premix and 0.05% of yttrium oxide; major component/%: 46.62 of crude protein, 11.83 of crude fat, 10.48 of crude ash, 2.78 of lysine content and 0.99 of methionine content.
Preparation of a spraying liquid: the spray solution, i.e., the lactic acid bacteria preparation prepared in example 3, was prepared as described above.
Spraying: 100 mu L of spraying liquid is taken by a liquid transfer gun in a clean bench and evenly sprayed into 1g of feed, and after the feed is naturally dried for 8 hours (the feed is observed to be dry by naked eyes) in the clean bench, the culture dish is sealed and put into a sealed bag and then put into a refrigerator at 4 ℃.
And (3) counting colonies: after the spraying is finished, the number of the lactic acid bacteria in the feed of each culture dish is counted by a colony counting method regularly.
The sample pretreatment method comprises the following steps: 1g of the feed was aseptically pulverized in a mortar, poured into a 15mL test tube containing 9mL of sterile water (1g of the feed was measured in volume of 1 mL), shaken for 2 minutes, and allowed to stand for layering, and the middle part of the supernatant was collected.
After the nano plant glycogen lactobacillus compound protective agent is preserved for 1 week, the survival rate of the lactobacillus in the feed is improved by more than 6 times. Therefore, the survival rate of the lactobacillus is obviously improved by the three-layer protective agent.
Influence of one or more protective agents on lactobacillus feed
Designing single-factor experiment groups, wherein the total number of the experiment groups is seven: blank group, skimmed milk powder 5%, skimmed milk powder 10%, skimmed milk powder 15%, trehalose 5%, trehalose 10%, trehalose 15%. Mixing the above materials with lactobacillus to make the concentration of lactobacillus about 109cfu/mL; then spraying micropterus salmoides feed with the spraying amount of 100 mu L/1g feed; the colony count of the lactobacillus in the feed is detected on the 2 nd, 4 th, 7 th and 12 th days after spraying respectively. The results of the experiment are shown in table 1:
TABLE 1 Effect of different concentrations of protective agent on the colony count of Lactobacillus feedstuff
Figure BDA0002486975530000081
a indicates significant difference from blank group, b indicates significant difference from the same column group marked with a (P <0.05)
As can be seen from Table 1, the groups added with 10% of milk powder on days 2, 4, 7 and 12 after spraying are all significantly different from the blank group, and the effect of adding 10% of milk powder is better than that of 15% of milk powder, but the trehalose at different concentrations has no significant protective effect on lactic acid bacteria, probably because of the individual difference of strains.
Secondly, the influence of adding water-soluble nano-phytoglycogen with different concentrations into 10 percent of skim milk powder protective agent on the colony count of the lactobacillus feed
Adding different water-soluble nano-phytoglycogen into 10% milk powder protective solution respectively to make the mass percentage of phytoglycogen in the protective solution respectively be 0%, 0.5%, 1% and 1.5%, then adding thallus into the obtained protective solution to make bacterial liquid concentration be about 109CFU/mL. The results of the experiment are shown in FIG. 5.
As can be seen from fig. 5, the addition of phytoglycogen at different concentrations increased colony survival rate, and 1% phytoglycogen significantly increased from the control group, and was consistently higher than 0.5% phytoglycogen group, and was comparable to 1.5% phytoglycogen group. Therefore, from the viewpoint of the survival rate of colonies of four groups and the saving of raw materials, the protective effect is best by adding 1% phytoglycogen. The nano-phytoglycogen is a highly branched carbohydrate, and is observed by a Transmission Electron Microscope (TEM) to be in a spherical particle with the diameter of 100-500 nm in an aqueous solution (figure 6). Because the nano particles are tiny in volume and good in water solubility, the nano particles are uniformly dispersed in the skim milk powder solution, and a compact second layer of protection is formed on lactobacillus casei cells.
Influence of Pickering emulsion added with hydrophobic nano-phytoglycogen with different concentrations in 10% of skimmed milk powder and 1% of water-soluble phytoglycogen mixed protective agent on colony count of lactobacillus feed
Adding 5%, 10% and 15% (w/w) of Pickering emulsion of hydrophobic nano-phytoglycogen into the second layer of protective solution, and homogenizing at 18000rpm for 4 minutes at high speed to form a third layer of nano-phytoglycogen lactic acid bacteria composite protective solution. Then adding thallus into the obtained protective solution to make the concentration of the bacterial solution about 109CFU/mL. The results are shown in FIG. 7.
As can be seen from fig. 7, the addition of Pickering emulsions of different concentrations of hydrophobic nano phytoglycogen increases the colony survival rate, and from day 3, the addition of Pickering emulsion of 10% hydrophobic nano phytoglycogen significantly increased compared to the control group, and was consistently significantly higher than the Pickering emulsion group of 5% hydrophobic nano phytoglycogen and better than the Pickering emulsion group of 15% hydrophobic nano phytoglycogen. Therefore, from the viewpoint of the colony survival rate of four groups and the saving of raw materials, the Pickering emulsion added with 10% of hydrophobic nano phytoglycogen has the best protection effect. The Pickering emulsion of hydrophobic nano phytoglycogen enables the distribution of bacteria liquid in skim milk powder and phytoglycogen protective solution to be more uniform, so that bacteria can be more fully wrapped by the protective solution; meanwhile, the Pickering emulsion of the hydrophobic nano phytoglycogen can enhance the oxidation stability of the bacterial liquid, so that the survival rate of the bacterial liquid is improved.
Influence of different protective agents on colony count of lactobacillus feed
Preparing different layers of protective agents, which respectively are as follows: a is a blank group without a protective agent; b is a single-layer protective solution, 10% of skimmed milk powder; c is 10% skimmed milk powder and 1% phytoglycogen two-layer protective solution; d is a Pickering emulsion composite protective solution of 10 percent of skimmed milk powder, 1 percent of phytoglycogen and 10 percent of hydrophobic nano-phytoglycogen. Adding the protective solution into thallus to make the concentration of the thallus about 109CFU/mL. The number of blank groups of colonies after the spraying was finished was determined as: 8.1351log CFU/g. The results are shown in FIG. 8.
As can be seen from fig. 8, the effect of the treatment group on the protection of lactic acid bacteria was superior to that of the blank group as a whole, and the difference was significant (P <0.05), and the effect of the group D was superior to that of the group B, C. From the results on day 7 of the deposit, it can be seen that group D is improved by more than 6 times compared to group a and 1.6 times compared to group B. Compared with the colony number after the spraying is finished, after the feed is preserved for 1 week, the survival rate of the colony number is 65.9 percent by adding the group D protective solution, while the survival rate of the group A is only 9.9 percent, so that the survival rate of the lactobacillus is remarkably improved by the group D protective solution. After water-soluble nano-phytoglycogen is added, the phytoglycogen nano-particles connect one milk ball to form a compact film to wrap the lactic acid bacteria. Then, after the Pickering emulsion of the hydrophobic nano phytoglycogen is added, the lactobacillus particles can be further dispersed and can be wound, so that a better protection effect is achieved.
Fifthly, evaluating the protection effect of the composite protective agent on the lactic acid bacteria powder under freeze drying
And (3) subpackaging 4mL of the bacterial suspension into sterile vials, pre-freezing the vials in a refrigerator at the temperature of-80 ℃ for 3 hours, and quickly transferring the vials to a freeze dryer for freeze drying after the bacterial suspension is completely frozen. The freeze-drying conditions were: vacuum degree of 10Pa, cold trap temperature of-55 deg.C, heating with a separator at 10 deg.C for 2h, heating to 20 deg.C, and lyophilizing for about 24 h.
Calculation of the freeze-drying survival rate:
(1) counting viable bacteria: and (3) rehydrating the freeze-dried bacterial powder, taking an MRS culture medium as a counting culture medium, and counting by adopting a gradient dilution plate method.
(2) Calculating the freeze-drying survival rate: the cell viability is the total viable count of the sample after lyophilization/the total viable count of the sample before lyophilization × 100%.
The results of the experiment are shown in table 2:
TABLE 2 influence of different protective solutions on the survival rate of lactic acid bacteria
Figure BDA0002486975530000101
Different letters indicate significant differences from the same column (P <0.05)
As can be seen from Table 2, the protective agent of group D is superior to that of group C, and significantly superior to that of groups B and A.
Sixthly, tolerance of artificial gastric juice
Preparing artificial simulated gastric juice: after 16.4mL of dilute hydrochloric acid of 0.1mol/L, 10.0g of pepsin and 500mL of water are added and shaken, water is added to the mixture until the volume is 1000mL for later use (pH 1.2).
0.2mL of cell suspension (10)9CFU/mL) (with different protective agents) is inoculated into 1.0mL artificial gastric juice (pH 2.0) and 0.3mL physiological saline which are subjected to membrane filtration sterilization (0.22 μm), fully and uniformly mixed, treated in a constant-temperature water bath at 37 ℃, sampled after the artificial gastric juice is treated for 1min, 90min and 180min respectively, and subjected to gradient dilution, and counted by a standard plate method (the dilution multiple is 10)-10) Three replicates. The results of the experiment are shown in table 3.
TABLE 3 different layer protective agent artificial gastric juice resistance
Figure BDA0002486975530000111
Different letters indicate significant differences from the same column (P <0.05)
As can be seen from Table 3, in the artificial gastric juice, the lactobacillus casei has no significant difference between each protective layer and the blank group within 1 minute, but has significant difference from the initial value, and after 90 minutes, the protective agents of each layer have significant difference from the blank group, which indicates that the protective agents of each layer have significant protective effect on the lactobacillus casei, but have no significant difference between the layers. After 180 minutes, each layer of protective agent has obvious protective effect on lactobacillus casei, and the protective effect of the three-layer protective agent is obviously higher than that of the two-layer protective agent and the single-layer protective agent.
Tolerance of artificial intestinal juice
Preparing artificial simulated intestinal juice: 6.8g of monopotassium phosphate is taken and dissolved by adding 500mL of water, and then adjusted to 6.8 by 0.1mol/L of sodium hydroxide. Then 10.0g of pancreatin is taken and dissolved by adding a small amount of water. Mixing the two solutions, adding water to a constant volume of 1000mL, and mixing uniformly for later use.
0.2mL of cell suspension (10)9CFU/mL) (with different protective agents) is inoculated into membrane filtration sterilized (0.22 μm) artificial small intestine solution (pH 8.0) and 0.3mL NaCl, mixed well, treated in a constant temperature water bath at 37 deg.C, sampled when the artificial small intestine solution is treated for 1min and 240min respectively, and counted by standard plate method (dilution multiple is 10) after gradient dilution-10) Three replicates. The results of the experiment are shown in table 4.
TABLE 4 different layer protectant Artificial intestinal juice tolerance
Figure BDA0002486975530000112
Different letters indicate significant differences from the same column (P <0.05)
As can be seen from table 4, in the artificial intestinal juice, the protective layers of lactobacillus casei are not significantly different from the blank group within 1 minute and are also not significantly different from the initial values, after 240 minutes, the protective agents of the respective layers are significantly different from the blank group and the initial values, and the protective effect of the protective agent of the three layers is significantly higher than that of the two layers and the single layer.
The foregoing is directed to preferred embodiments of the present invention, other and further embodiments of the invention may be devised without departing from the basic scope thereof, and the scope thereof is determined by the claims that follow. However, any simple modification, equivalent change and modification of the above embodiments according to the technical essence of the present invention are within the protection scope of the technical solution of the present invention.

Claims (7)

1. A nano phytoglycogen lactobacillus composite protective agent is characterized by comprising skimmed milk powder, water-soluble nano phytoglycogen, Pickering emulsion of hydrophobic nano phytoglycogen and normal saline;
wherein the content of the first and second substances,
the content of the skimmed milk powder in the composite protective agent is 5-15% w/w;
the content of the water-soluble nano phytoglycogen in the composite protective agent is 0.5-1.5% w/w;
the addition amount of the Pickering emulsion of the hydrophobic nano phytoglycogen in the composite protective agent is 5-15% w/w;
the balance of the raw materials is normal saline water,
the water-soluble nano phytoglycogen is prepared by the following method:
weighing sweet corn seeds, adding deionized water, and soaking at 4 ℃ for 24 h; removing the water solution from the soaked corn seeds, crushing, and then mixing the corn seeds according to the proportion of 1: adding deionized water at a material-water ratio of 3v/v, and standing for 24h at 4 ℃; taking the supernatant, adding acetic acid to adjust the pH to 4.8-5, and standing at 4 deg.C for 24 hr; centrifuging at 3000g for 10min to remove precipitate, boiling the obtained supernatant, centrifuging at 3000g for 10min again to further remove protein until no oil exists and no precipitate exists; adding absolute ethyl alcohol into the obtained supernatant according to the volume ratio of 1:1, stirring and mixing, standing at 4 ℃ until the precipitate is completely precipitated, centrifuging 3000g, taking the precipitate, and freeze-drying to obtain the water-soluble nano phytoglycogen and the preparation method of the Pickering emulsion of the hydrophobic nano phytoglycogen, wherein the preparation method comprises the following steps: mixing the hydrophobic nano-phytoglycogen with deionized water until the concentration is 5% w/w, adding medium-chain triglyceride to 50% w/w, homogenizing at a high speed of 18000rpm for 4 minutes at a pH value of 7.0 to obtain a Pickering emulsion of the hydrophobic nano-phytoglycogen, wherein the preparation method of the hydrophobic nano-phytoglycogen comprises the following steps: diluting octenyl succinic anhydride to 20% w/w in isopropanol, adding into 30% w/w water-soluble nano-phytoglycogen suspension, and diluting to 10% w/w water-soluble nano-phytoglycogen suspension; adjusting the pH value to 8.5 by using a 3% NaOH solution, fully reacting for 2 hours, and adjusting the pH value to 6.5 by using 2.5M hydrochloric acid; keeping the temperature at 35 ℃ for 8 hours, centrifuging 3000g for 10 minutes after the culture is finished, taking the precipitate, washing the precipitate by distilled water and absolute ethyl alcohol in sequence, and repeating the steps for 3 times to obtain the hydrophobic nano-phytoglycogen.
2. The method for preparing the nano-phytoglycogen lactic acid bacteria compound protective agent as claimed in claim 1, characterized by comprising the following steps:
(1) mixing skimmed milk powder with 0.85% of autoclaved normal saline, and shaking and mixing to obtain a first layer of protective agent;
(2) adding the sterilized and freeze-dried water-soluble nano phytoglycogen into the first layer of protective agent, and uniformly oscillating to form a compact second layer of protective solution;
(3) adding Pickering emulsion of hydrophobic nano-phytoglycogen into the second layer of protective solution, adjusting the pH to 7, homogenizing at 18000rpm for 4 minutes at high speed, and forming a third layer of nano-phytoglycogen lactic acid bacteria composite protective solution to obtain the nano-phytoglycogen lactic acid bacteria composite protective agent.
3. The method for preparing the nano-phytoglycogen lactic acid bacteria compound protectant according to claim 2, wherein the nano-phytoglycogen lactic acid bacteria compound protectant is characterized in that,
the content of the skimmed milk powder in the composite protective agent is 10% w/w;
the content of the water-soluble nano phytoglycogen in the composite protective agent is 1% w/w;
the addition amount of the Pickering emulsion of the hydrophobic nano phytoglycogen in the composite protective agent is 10% w/w.
4. The use of the nano-phytoglycogen lactic acid bacteria compound protectant prepared by the method of claim 3 in preparing a lactic acid bacteria preparation is characterized by being used for improving the survival rate of lactic acid bacteria and the tolerance of lactic acid bacteria to gastric juice and intestinal juice.
5. A lactobacillus preparation, characterized in that lactobacillus mud obtained by fermentation and centrifugation is added into the nano-phytoglycogen lactobacillus composite protective agent prepared by the method of claim 3, and the mixture is uniformly mixed to ensure that the concentration of the lactobacillus is 109cfu/mL。
6. Use of the lactic acid bacteria preparation according to claim 5 for the preparation of a lactic acid bacteria feed.
7. A fish feed containing lactic acid bacteria, characterized in that the lactic acid bacteria preparation according to claim 5 is sprayed on a fish feed to obtain the fish feed containing lactic acid bacteria; wherein the spraying amount of the nano plant glycogen lactobacillus composite protective agent is 100 mu L/g feed.
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