CN113862202B - Method for promoting lactic acid bacteria proliferation by using Maillard reaction product - Google Patents
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Abstract
The invention relates to a method for promoting lactobacillus proliferation by using Maillard reaction products, which comprises the following steps: and adding the Maillard reaction product SMS-MRPs dry powder into an MRS liquid culture medium or a GM17 liquid culture medium according to the addition amount of 0.5-15.0 mg/mL, and fully dissolving to realize the proliferation of the lactic acid bacteria. The preparation method has the advantages of simple operation steps, higher preparation efficiency and lower production cost.
Description
Technical Field
The invention relates to the technical field of bioengineering, in particular to a method for promoting lactobacillus proliferation by using Maillard reaction products.
Background
The human intestinal tract gathers complex and diverse bacteria, and the imbalance of intestinal flora can cause inflammatory bowel disease, colorectal cancer and other metabolic diseases. A large number of clinical trials show that the intake of probiotics and prebiotics has the function of preventing and treating various common diseases of human bodies. However, the probiotic bacteria need to overcome gastric acid, bile salts and various intestinal digestive enzymes when entering the intestinal tract to exert the probiotic effect, and the probiotic bacteria have the characteristics of high survival rate and strong planting force. In contrast, a prebiotic is a non-digestible compound that modulates the composition and/or activity of the intestinal flora through metabolism of the intestinal microorganisms, thereby producing a beneficial physiological effect on the host. Therefore, the development and application of prebiotics is more universal.
Currently, the main components of the prebiotic materials that are proven are non-digestible carbohydrates, such as functional oligosaccharides, polysaccharides and natural plant extracts. The study by Sarikaya et al found that inulin could be fermented by intestinal microbial suspensions to promote the growth of lactobacilli and bifidobacteria. In addition, he Jun and other researches show that the fructo-oligosaccharide and the inulin can slow down the decay of bifidobacteria in the fermented milk, and the inulin has better effect. Studies such as Szotysik show that the dry powder of the rose extract can improve the content of probiotics in the yoghurt. These sugars can be bound, taken up or utilized by the transporters or enzymes specifically expressed by the probiotics and thus can selectively promote the growth of the probiotics. However, most of the existing prebiotic materials do not contain a protein source. Studies have shown that proteins and certain amino acids are important nitrogen sources and growth factors for the intestinal flora, since most of the ingested proteins are absorbed by the small intestine, resulting in a deficiency of amino acids in the colon, thus limiting the proliferation of probiotics. Thus, the development of protein-derived prebiotic materials capable of reaching the colon for use by colonic microorganisms is a new direction of prebiotic research.
Studies have shown that casein and its polypeptides, lactoferrin and its polypeptides, etc. all significantly promote the growth of bifidobacteria and lactobacilli, and that degradation of milk proteins can produce amino acids such as leucine, cysteine, tryptophan, tyrosine, etc. necessary for the growth of probiotics. The digestibility of protein in stomach and intestine is obviously reduced after the protein is modified by Maillard reaction glycosylation, and most MRPs can not be absorbed by front digestive system but can only be utilized by colon microorganisms in a glycolysis or proteolysis mode, which shows that protein glycosylation is a modification means for effectively improving the stomach and intestine passing rate of protein. However, the Maillard reaction is affected by the concentration of reducing sugar, temperature and time. Meanwhile, fat oxidation or decomposition of milk fat occurs during the heat treatment, and thus the maillard reaction degree may be adversely affected.
Disclosure of Invention
The invention aims to provide a method for promoting the proliferation of lactic acid bacteria by utilizing Maillard reaction products, which has low cost and high protein utilization rate.
In order to solve the above problems, the method for promoting the proliferation of lactic acid bacteria by using Maillard reaction products of the invention is characterized in that: and adding the Maillard reaction product SMS-MRPs dry powder into an MRS liquid culture medium or a GM17 liquid culture medium according to the addition amount of 0.5-15.0 mg/mL, and fully dissolving to realize the proliferation of the lactic acid bacteria.
The SMS-MRPs dry powder is prepared by the following method: degreasing fresh raw milk to obtain defatted milk, and regulating the pH of the defatted milk to 8.0-9.5; then adding glucose into the defatted milk until the concentration is 6.0 g/100mL, and carrying out Maillard reaction at 95 ℃ for 120 min to obtain a reaction solution; the reaction solution is prepared by cooling with ice water, rotary evaporation and freeze drying.
The degreasing milk is prepared by filtering fresh raw milk by 4 layers of gauze to obtain filtrate, and centrifuging the filtrate for 15-30 min at the temperature of 4 ℃ and under the condition of 4000-5500 r/min.
The characteristic absorbance value of the reaction liquid is 0.827-0.863.
The condition of rotary evaporation is that the temperature is 45-60 ℃, the rotating speed is 40-55 r/min, and the time is 25-40 min.
The freeze drying condition means that the temperature is below-50 ℃ and the time is 20-26 hours.
The MRS liquid culture medium is prepared by sequentially adding 10 g peptone, 8 g beef extract, 5 g yeast extract powder, 20 g glucose, 2 g citric acid diamine, 5 g sodium acetate, 2 g dipotassium hydrogen phosphate, and 0.58 g MgSO into 1000 mL distilled water 4 ·7H 2 O、0.25 g MnSO 4 ·4H 2 And (3) mixing the mixture with O, 1 mL Tween 80 and stirring uniformly to obtain the finished product.
The GM17 liquid cultureThe basic method comprises sequentially adding 5 g fish peptone, 5 g tryptone, 2.5 g yeast extract powder, 5 g beef extract, 3 g lactose, 0.5 g sodium ascorbate and 0.25 g MgSO into 1000 mL distilled water 4 ·7H 2 And (3) uniformly stirring the O and the 5 g potassium dihydrogen phosphate.
Compared with the prior art, the invention has the following advantages:
1. the SMS-MRPs dry powder prepared by the invention can be used as a protein source prebiotic substance for enrichment culture of lactobacillus and the like, can promote proliferation and growth speed of lactobacillus to different degrees under the addition of 0.5-15.0 mg/mL, and can obviously improve the acid production capacity of streptococcus thermophilus.
2. The SMS-MRPs dry powder produced by the invention takes raw milk as raw material, has wide sources and stable yield.
3. The method has the advantages of simple operation steps, higher preparation efficiency and lower production cost, and can increase the utilization rate of colon microorganisms on the cow milk protein.
Drawings
The following describes the embodiments of the present invention in further detail with reference to the drawings.
FIG. 1 shows the effect of different addition amounts of SMS-MRPs prepared by the invention on the growth characteristics of lactobacillus bacteria liquid. Wherein: a is lactobacillus paracasei L9; b is lactobacillus rhamnosus G5; c is Lactobacillus reuteri G8; d is Streptococcus thermophilus Q2.
FIG. 2 shows the effect of different addition amounts of SMS-MRPs prepared by the invention on dry weight of four strains of lactobacillus. And (3) injection: the different letters represent obvious differencep<0.05)。
FIG. 3 shows the effect of SMS-MRPs prepared according to the invention on the acid producing ability of lactic acid bacteria. Wherein: a is lactobacillus paracasei L9; b is lactobacillus rhamnosus G5; c is Lactobacillus reuteri G8; d is Streptococcus thermophilus Q2. And (3) injection: * Representing that the difference between different treatment groups at the same time is very remarkableP<0.01）。
Detailed Description
A method for promoting proliferation of lactobacillus by using Maillard reaction products comprises the steps of adding dry powder of the Maillard reaction products SMS-MRPs into MRS liquid culture medium or GM17 liquid culture medium with the addition amount of 0.5-15.0 mg/mL, and fully dissolving to realize proliferation of lactobacillus.
Wherein:
the SMS-MRPs dry powder is prepared by the following method: degreasing fresh raw milk: filtering fresh raw milk by 4 layers of gauze to obtain a filtrate, centrifuging the filtrate for 15-30 min at the temperature of 4 ℃ and under the condition of 4000-5500 r/min to obtain defatted milk, and regulating the pH of the defatted milk to 8.0-9.5. And then adding glucose into the skimmed milk until the concentration is 6.0 g/100mL, and performing Maillard reaction at 95 ℃ for 120 min to obtain a reaction solution, wherein the characteristic light absorption value of the reaction solution is 0.827-0.863. And cooling the reaction liquid by ice water, and performing rotary evaporation at the temperature of 45-60 ℃ and the speed of 40-55 r/min for 25-40 min to obtain a concentrated solution, and performing freeze drying on the concentrated solution for 20-26 h at the temperature of below-50 ℃.
MRS liquid culture medium is prepared by sequentially adding 10 g peptone, 8 g beef extract, 5 g yeast extract powder, 20 g glucose, 2 g diamine citrate, 5 g sodium acetate, 2 g dipotassium hydrogen phosphate, 0.58 g MgSO into 1000 mL distilled water 4 ·7H 2 O、0.25 g MnSO 4 ·4H 2 And (3) mixing the mixture with O, 1 mL Tween 80 and stirring uniformly to obtain the finished product.
The GM17 liquid culture medium is prepared by sequentially adding 5 g fish peptone, 5 g tryptone, 2.5 g yeast extract, 5 g beef extract, 3 g lactose, 0.5 g sodium ascorbate, and 0.25 g MgSO into 1000 mL distilled water 4 ·7H 2 And (3) uniformly stirring the O and the 5 g potassium dihydrogen phosphate.
Example 1
Weighing 500 g fresh raw milk, filtering with 4 layers of gauze to obtain filtrate, centrifuging and degreasing the filtrate at 5500 r/min for 15 min to obtain skimmed milk. Regulating the pH of the skim milk to 9.5 by using a NaOH solution with the concentration of 1 mol/L; glucose is added into the defatted milk until the concentration is 6.0 g/100mL, and the mixture is placed into a water bath at 95 ℃ to react for 120 min so as to fully generate Maillard reaction, thus obtaining reaction liquid. After the reaction is finished, cooling the reaction solution by ice water, then transferring the reaction solution into a round bottom flask, and performing rotary evaporation for 25 min at 60 ℃ under the condition of 55 r/min to obtain concentrated solution; adding the concentrated solution into a freeze dryer, maintaining the temperature below-50 ℃, and freeze-drying 26-h to obtain SMS-MRPs dry powder.
Example 2
Weighing 500 g fresh raw milk, filtering with 4 layers of gauze to obtain filtrate, centrifuging at 5000 r/min, and degreasing for 20 min to obtain skimmed milk. Regulating the pH of the skim milk to 9.0 by using a NaOH solution with the concentration of 1 mol/L; glucose is added into the defatted milk until the concentration is 6.0 g/100mL, and the mixture is placed into a water bath at 95 ℃ to react for 120 min so as to fully generate Maillard reaction, thus obtaining reaction liquid. After the reaction is finished, cooling the reaction solution by ice water, transferring the reaction solution into a round bottom flask, and performing rotary evaporation for 30 min at 55 ℃ under the condition of 50 r/min to obtain concentrated solution; adding the concentrated solution into a freeze dryer, keeping the temperature below-50 ℃, and freeze-drying 24-h to obtain SMS-MRPs dry powder.
Example 3
Weighing 500 g fresh raw milk, filtering with 4 layers of gauze to obtain filtrate, centrifuging and degreasing the filtrate at 4500 r/min for 25 min to obtain skimmed milk. Regulating the pH of the skim milk to 8.5 by using a NaOH solution with the concentration of 1 mol/L; glucose is added into the defatted milk until the concentration is 6.0 g/100mL, and the mixture is placed into a water bath at 95 ℃ to react for 120 min so as to fully generate Maillard reaction, thus obtaining reaction liquid. After the reaction is finished, cooling the reaction solution by ice water, transferring the reaction solution into a round bottom flask, and performing rotary evaporation for 35 min at 50 ℃ under the condition of 45 r/min to obtain concentrated solution; adding the concentrated solution into a freeze dryer, maintaining the temperature below-50 ℃, and freeze-drying 22-h to obtain SMS-MRPs dry powder.
Example 4
Weighing 500 g fresh raw milk, filtering with 4 layers of gauze to obtain filtrate, centrifuging at 4000 r/min, and degreasing for 30 min to obtain skimmed milk. Regulating the pH of the skim milk to 8.0 by using a NaOH solution with the concentration of 1 mol/L; glucose is added into the defatted milk until the concentration is 6.0 g/100mL, and the mixture is placed into a water bath at 95 ℃ to react for 120 min so as to fully generate Maillard reaction, thus obtaining reaction liquid. After the reaction is finished, cooling the reaction solution by ice water, transferring the reaction solution into a round bottom flask, and performing rotary evaporation for 40 min at 45 ℃ under the condition of 40 r/min to obtain concentrated solution; adding the concentrated solution into a freeze dryer, keeping the temperature below-50 ℃, and freeze-drying 20-h to obtain SMS-MRPs dry powder.
The SMS-MRPs dry powders obtained in examples 1-4 were added to MRS or GM17 liquid medium at different concentrations (0.5 mg/mL, 0.9 mg/mL, 1.8 mg/mL, 3.7 mg/mL, 7.5 mg/mL and 15.0 mg/mL) and dissolved well. And respectively transferring 10 mu L of lactobacillus bacterial liquid and 250 mu L of MRS or GM17 liquid culture medium to the ELISA plate, culturing at 37 ℃ for 24 h, and taking bacterial liquid every 2 h to detect OD values. MRS or GM17 broth without SMS-MRPs dry powder was used as a control. The results are shown in FIGS. 1-3 and Table 1.
TABLE 1 SMS-MRPs against four strains of lactic acid bacteria viable count (10) 8 CFU/mL) effect
As can be seen from fig. 1 to 3, the OD value of the SMS-MRPs treated group is significantly increased as compared with the control group along with the increase of the addition amount of the SMS-MRPs, which indicates that the SMS-MRPs have excellent bacteria increasing effect, so that the growth and metabolism ability of the lactic acid bacteria can be promoted to different degrees after the lactic acid bacteria are degraded and utilized by the SMS-MRPs. And can remarkably promote the acid production capacity of streptococcus thermophilus Q2P<0.01）。
As can be seen from Table 1, after 15.0. 15.0 mg/mL of SMS-MRPs are added, the viable count of lactic acid bacteria is significantly increasedP<0.05）。
Claims (5)
1. A method for promoting lactobacillus proliferation by using Maillard reaction products, which is characterized by comprising the following steps: adding the Maillard reaction product SMS-MRPs dry powder into an MRS liquid culture medium or a GM17 liquid culture medium according to the addition amount of 0.5-15.0 mg/mL, and fully dissolving to realize the proliferation of lactic acid bacteria; the SMS-MRPs dry powder is prepared by the following method: degreasing fresh raw milk to obtain defatted milk, and regulating the pH of the defatted milk to 8.0-9.5; then adding glucose into the defatted milk until the concentration is 6.0 g/100mL, and carrying out Maillard reaction at 95 ℃ for 120 min to obtain a reaction solution; the reaction solution is prepared by cooling with ice water, rotary evaporation and freeze drying; the MRS liquid culture medium is prepared by sequentially adding 10 g peptone, 8 g beef extract, 5 g yeast extract powder, 20 g glucose, 2 g citric acid diamine, 5 g sodium acetate, 2 g dipotassium hydrogen phosphate, and 0.58 g MgSO into 1000 mL distilled water 4 ·7H 2 O、0.25 g MnSO 4 ·4H 2 Stirring with O, 1 mL Tween 80Uniformly stirring to obtain the product; the GM17 liquid culture medium is prepared by sequentially adding 5 g fish peptone, 5 g tryptone, 2.5 g yeast extract, 5 g beef extract, 3 g lactose, 0.5 g sodium ascorbate and 0.25 g MgSO into 1000 mL distilled water 4 ·7H 2 And (3) uniformly stirring the O and the 5 g potassium dihydrogen phosphate.
2. A method for promoting lactic acid bacteria proliferation using maillard reaction products as in claim 1 wherein: the degreasing milk is prepared by filtering fresh raw milk by 4 layers of gauze to obtain filtrate, and centrifuging the filtrate for 15-30 min at the temperature of 4 ℃ and under the condition of 4000-5500 r/min.
3. A method for promoting lactic acid bacteria proliferation using maillard reaction products as in claim 1 wherein: the characteristic absorbance value of the reaction liquid is 0.827-0.863.
4. A method for promoting lactic acid bacteria proliferation using maillard reaction products as in claim 1 wherein: the condition of rotary evaporation is that the temperature is 45-60 ℃, the rotating speed is 40-55 r/min, and the time is 25-40 min.
5. A method for promoting lactic acid bacteria proliferation using maillard reaction products as in claim 1 wherein: the freeze drying condition means that the temperature is below-50 ℃ and the time is 20-26 hours.
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| CN110477101A (en) * | 2019-08-28 | 2019-11-22 | 江南大学 | A kind of brown Yoghourt and preparation method thereof of low 5 hydroxymethyl furfural content |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110477101A (en) * | 2019-08-28 | 2019-11-22 | 江南大学 | A kind of brown Yoghourt and preparation method thereof of low 5 hydroxymethyl furfural content |
Non-Patent Citations (3)
| Title |
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| Formation and Alterations of the Potentially Harmful Maillard Reaction Products during the Production and Storage of Brown Fermented Milk;Zhonghui Han等;Molecules;第24卷(第2期);272 * |
| 发酵凝乳菌种的优化及美拉德反应时间对灭菌型乳酸菌饮料稳定性的影响;赵晶等;食品安全质量检测学报;第8卷(第12期);4539-4546 * |
| 热处理对牛乳美拉德反应程度及挥发性成分的影响;依胜男等;食品科学;第42卷(第14期);9-15 * |
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