TWI737210B - Starter culture protection formula and use thereof - Google Patents

Abstract

A starter culture protection formula includes 1.125±10% to 4.5±10% by weight of trehalose; 2.25±10% to 4.5±10% by weight of skim milk powder; 1.125±10% to 2.25±10% by weight of mannitol; 0.0225±10% to 0.1125±10% by weight of Vitamin C; 0.225±10% to 1.125±10% by weight of monosodium glutamate; and 0.225±10% to 1.125±10% by weight of glycerol.

Description

Bacteria protective agent formula and its use

The present invention relates to a bacterial protective agent formula, especially suitable for both Gram-positive bacteria and Gram-negative bacteria.

Since the number of natural microorganisms usually does not reach the number required for actual use, and it is very difficult to preserve, they are often stimulated by the environment or produce mutations after multiple generations of culture, so they are mostly processed by hot air drying or freeze drying. "Bacteria" (starter) type for use and circulation. Take the food industry as an example. Due to its large volume of operations, if microorganisms are required for fermentation in the manufacturing process, natural bacteria cannot be used directly. The domesticated bacteria are often used to expand the culture first, and then the follow-up process can be carried out after reaching a sufficient number of bacteria. The actual industrial applications include industries that require yeast as a raw material, such as the fermentation of beer and the baking industry of bread. In addition, as applied to the agricultural biologics industry, the biologics products produced also face the requirement of high and stable viable counts, so it is also suitable for the first expansion of bacteria. Therefore, after discovering the characteristic potential strains, a kind of bacterial formula is needed, which can achieve the purpose of high stability and high viable cell count, so as to increase the application flexibility in the manufacturing process. At the same time, the bacteria can be sold in the form of bacteria, increasing the speed and diversity of product innovation.

CN106244459A discloses a pseudomonas dry powder and a preparation method thereof. The pseudomonas dry powder comprises pseudomonas and a protective agent, wherein the protective agent includes skimmed milk powder, mannitol, sodium glutamine or glycine, corn starch And trehalose.

CN107828683A discloses a Lactobacillus plantarum freeze-dried powder for extending the shelf life of yogurt and a preparation method. The Lactobacillus plantarum freeze-dried powder is prepared by mixing and freeze-drying the Lactobacillus plantarum strain and the emulsification protective agent, wherein the Lactobacillus plantarum strain is stored in In the China General Microbial Culture Collection Center (CGMCC), the deposit number is CGMCC NO. 10453. The preparation method includes the steps of strain activation, strain propagation, fermentation culture and freeze-drying. The freeze-dried Lactobacillus plantarum powder has an inhibitory effect on the main spoilage molds and yeasts in fermented dairy products.

The current formula of bacterial protection agent has the disadvantage that the number of viable bacteria is low during culture. Especially when the formula of bacterial protection agent is used for a period of time after the preparation is completed, the number of viable bacteria is greatly reduced.

The main purpose of the present invention is to provide a formula of bacterial protection agent, which can improve the shortcomings of the number of viable bacteria and low survival rate, especially a formula of bacterial protection agent with improved stability. The prepared bacterial protection agent formula still has a comparable number of viable bacteria.

According to the purpose of the present invention, a formula of a protective agent for bacterial decay includes: 1.125±10% by weight of trehalose; 2.25±10% by weight of skimmed milk powder; 1.125±10% by weight of mannitol; 0.0225±10% by weight of vitamin C; 0.225±10% by weight of sodium glutamate; and 0.225±10% by weight of glycerin.

Other preferred embodiments of the present invention include but are not limited to those described in the scope of the following patent applications.

The formula of the bacterial protection agent of the present invention has a feature that no matter it is used for the culture of gram-positive bacteria or gram-negative bacteria, it has an improved number of viable bacteria and survival rate.

The present invention and its improvements will be further understood by the following examples and comparative examples. The following materials and equipment are used in the following examples and comparative examples. Material Skimmed milk powder MERCK Casein MERCK glycerin SIGMA Vitamin C SIGMA Trehalose SIGMA sucrose SIGMA Sodium Glutamate (MSG) Weiquan Span 60 SIGMA YM Broth Gibico Pseu F agar medium formula Trypsin 1% MERCK Trypsin 1% MERCK Dipotassium hydrogen phosphate (K ₂ HPO ₄ ) 0.15% SIGMA Magnesium sulfate (MgSO ₄ ) 0.15% SIGMA Agar SIGMA instrument Desktop centrifuge Centrifuge 5810R, eppendorf Floor-standing centrifuge Himac CR 21E Beckman J2-21M/E Centrifuge Freeze dryer SP Scientific#50L Virtual EL-85° Dura-Stop system Spectrophotometer V-550 UV/VIS Spectrophotometer, JASCO Fermentation equipment system Biotop Process & Equipment

Pseudomonas fluorescens culture method

Pseudomonas fluorescens YLW01 ( Pseudomonas putida ) was selected as the experimental strain. YLW01 culture containing 1% Molasses, 1% MSG, 1.5 g / LK 2 HPO 4 and ^{_{1.5 g / L MgSO 4. 7H}} 2 O of the medium (50 mL medium / 250 mL flask) at 30 ℃, 150 rpm culture 42 hr. After the culture is completed, centrifuge at 8000 rpm at 4°C for 10 minutes, discard the supernatant, wash with water and centrifuge again, and repeat this washing method twice. Afterwards, the centrifuged bacterial sludge was taken out and stored in the refrigerator.

Yeast culture method

Yeasts 21849, 20405, and 22745 were selected as experimental strains. Yeasts 21849, 20405, and 22745 were cultured in YM broth (Becton Dickinson and Company) medium (50 mL medium/250 mL Erlenmeyer flask), and cultured at 25°C and 250 rpm for 48 hr. After the culture is completed, centrifuge at 8000 rpm at 4°C for 10 minutes, discard the supernatant, wash with water and centrifuge again, and repeat this washing method twice. Afterwards, the centrifuged bacterial sludge was taken out and stored in the refrigerator.

Preservative formula/preparation of protective liquid

The protective solution is prepared by dissolving the protective agent formula in Table 1 in 22.5 mL of water, and stirring for 30 minutes until it is uniformly dissolved, then the following phosphate solution (adjusted from alkaline to acidic) or glycine-sodium hydroxide buffer The pH of the liquid (adjusted from acidic to alkaline) is adjusted to 7 or 8. Table 1: Protective agent formula (g)/protective solution (g/vol %) Protection fluid group pH value Trehalose (g) or (g/vol %) Skimmed milk powder (g) or (g/vol %) Mannitol (g) or (g/vol %) Vitamin C (g) or (g/vol %) Sodium glutamate (MSG) (g) or (g/vol %) Glycerin (g) or (g/vol %) 1 7 1.125 (5%) 2.25 (10%) 1.125 (5%) 0.0225 (0.1%) 0.225 (1%) 0.225 (1%) 2 7 1.125 (5%) 2.25 (10%) 2.25 (10%) 0.1125 (0.5%) 1.125 (5%) 1.125 (5%) 3 7 4.5 (20%) 4.5 (20%) 1.125 (5%) 0.0225 (0.1%) 1.125 (5%) 1.125 (5%) 4 7 4.5 (20%) 4.5 (20%) 2.25 (10%) 0.1125 (0.5%) 0.225 (1%) 0.225 (1%) 5 8 1.125 (5%) 4.5 (20%) 1.125 (5%) 0.1125 (0.1%) 0.225 (1%) 1.125 (5%) 6 8 1.125 (5%) 4.5 (20%) 2.25 (10%) 0.0225 (0.1%) 1.125 (5%) 0.225 (1%) 7 8 4.5 (20%) 2.25 (10%) 1.125 (5%) 0.1125 (0.5%) 1.125 (5%) 0.225 (1%) 8 8 4.5 (20%) 2.25 (10%) 2.25 (10%) 0.0225 (0.1%) 0.225 (1%) 1.125 (5%) Preparation of Glycine-Sodium Hydroxide Buffer: 50 mL of glycine aqueous solution (0.2M) and 8.8 mL of sodium hydroxide aqueous solution (0.2M) are mixed and quantified to 200 mL. Phosphate solution preparation: 87.7 mL sodium dihydrogen phosphate aqueous solution (0.2M) and 12.3 mL sodium monohydrogen phosphate aqueous solution (0.2M) are mixed uniformly and quantified to 200 mL. Example 1

Bacteria preparation

Take out the above-prepared bacterial sludge from the refrigerator, mix 7.5 g of bacterial sludge with each protection solution in Table 1, and stir for 30 minutes until the mixture is uniform, pre-freeze at -20°C and freeze-dry to obtain bacterial sludge.

Re-dissolve 1 g of the freshly prepared freeze-dried broccoli into 10 mL of water, and perform serial dilutions at an appropriate rate. Take 100 μL of the broccoli dilution and smear it on the Pseu F medium, and place it at 30°C for 72 Count the colonies after 1 hour incubation. Comparative example 1

The formula of the aforementioned CN106244459 and CN107828683 was used for the preparation of the bacteria. Take 22.5 mL of the protection solution in Table 2 and Table 3 and mix with 7.5 g of the bacterial paste prepared above, stir for 30 minutes until the mixture is uniform, pre-freeze at -20°C and freeze-dry to obtain the bacterial sludge of Comparative Example 1.

Re-dissolve 1 g of the freshly prepared lyophilized broccoli into 10 mL of water, and perform serial dilutions at an appropriate rate. Take 100 μL of the broccoli dilution and smear it on the Pseu F medium, and place it at 30°C for 72 Count the colonies after 1 hour incubation. Table 2 CN106244459 protection solution (g/vol %) Group Trehalose (%) Skimmed milk powder (%) Mannitol (%) yeast(%) MSG (%) Span60(%) Glycine (%) corn starch(%) CN106244459-1 1 25 0.5 1 3 2 3 0.1 CN106244459-2 5 12 5 3 0 1 15 2 Table 3 CN107828683 protection liquid (g/vol %) Group Trehalose (%) Skimmed milk powder (%) glycerin(%) Mannitol (%) sucrose(%) ascorbic acid water CN107828683 6 8 1 2 4 0.5 78.5

result

Fig. 1 shows the number of viable bacteria of Pseudomonas fluorescens bacteria prepared with each protection solution group in Table 1 of Example 1 after culturing at 30°C for 72 hours. It can be found in Fig. 1 that the number of viable Pseudomonas fluorescens is maintained above 10 ⁹ CFU/g, which shows that the protective agent of the present invention has a good protective effect. Among them, group 2 has the highest number of viable bacteria up to 6.3x10 ⁹ CFU/g.

Figures 2 to 4 respectively show the number of viable cells of yeasts 21849, 20405, and 22745 prepared with the protection liquid groups in Table 1 of Example 1 after culturing at 25°C for 72 hours. Yeast 21849, 20405, and 22745 had the highest viable counts of 3.44x10 ⁹ CFU/g (Figure 2 group 5), 3.75x10 ⁹ CFU/g (Figure 3 group 7) and 5x10 ⁹ CFU/g (Figure 4). Group 3).

Fig. 5 shows the number of viable bacteria of Pseudomonas fluorescens bacteria prepared with each protection solution group in Table 2 and Table 3 of Comparative Example 1 after culturing at 30°C for 72 hours. In Figure 5, it can be found that the number of viable Pseudomonas fluorescens is 5.8× ^{10 7} , 8.8× ^{10 8} and 2.6× ^{10 8} CFU/g, respectively.

Fig. 6 shows the number of viable cells of yeast 21849, 20405, and 22745 prepared by the protective liquid groups in Table 2 and Table 3 of Comparative Example 1, after culturing at 25°C for 72 hours. The culture effect was not good. Most of the viable bacteria count was 10 ⁸ CFU/g, and the group with the highest viable count was yeast 22745 using the formula of CN106244459-2, which reached 1.99x10 ⁹ CFU/g.

Fig. 7 shows the survival rate of Pseudomonas fluorescens bacterium prepared with each protection solution group in Table 1 of Example 1 after culturing at 30°C for 72 hours. The survival rate calculation method is as follows: Survival rate (%) = [Number of viable bacteria after lyophilization (CFU) / Number of bacteria added before lyophilization (CFU)]*100% It can be found in Figure 7 that the number of viable Pseudomonas fluorescens is maintained above 15%, and group 2 has the highest survival rate of about 78%. The performance of the survival rate of each group in Figure 7 is similar to the performance of the number of viable bacteria in Figure 1.

Figures 8 to 10 respectively show the survival rates of yeasts 21849, 20405, and 22745 prepared with the protective liquid groups in Table 1 of Example 1 after being cultured at 25°C for 72 hours. Yeast 21849, 20405, and 22745 had the highest survival rates of 53.22% (Figure 8 Group 3), 47.2% (Figure 9 Group 3) and 47.41% (Figure 10 Group 3).

Fig. 11 shows the survival rate of Pseudomonas fluorescens bacteria prepared in each protective solution group in Table 2 and Table 3 of Comparative Example 1 after culturing at 30°C for 72 hours. In Figure 11, it can be found that the survival rates of Pseudomonas fluorescens are 0.07%, 1.02%, and 0.3%, respectively.

Fig. 12 shows the survival rates of yeasts 21849, 20405, and 22745 prepared by the protective liquid groups in Table 2 and Table 3 of Comparative Example 1 after culturing at 25°C for 72 hours. The survival rate shown in Figure 12 has the same trend as the number of viable bacteria in Figure 6, about 2%, and the highest group is the culture of CN106244459-1 in yeast 20405.

It can be seen from Figures 1 to 12 above that the formulation of the present invention and the previous formulation of the formulation can be found by using Pseudomonas fluorescens and 3 kinds of yeasts (21849, 20405, 22745) to test the formulation of the present invention and the previous formulation. Compared with the previous formula CN106244459-1, CN106244459-2 and CN107828683, the number of viable bacteria and the survival rate are significantly improved. It is proved that the present invention has unexpected enhancement effect. Example 2: Stability Test of Lyophilized Bacteria

After storing the freeze-dried mushroom prepared in Example 1 at 25°C and 54°C for 14 days, re-dissolve 1 g of the bacteria into 10 mL of water, and perform serial dilutions of appropriate magnification, and take 100 μL of fluorescent false Smear the diluted solution of the monosporum bacteria on the Pseu F medium, place it at 30°C for 72 hours, and then count the colonies.

The results are shown in Fig. 13, which also shows the number of viable cells of the freshly prepared freeze-dried mushrooms. Figure 13 shows the Pseudomonas fluorescens bacterium prepared with each protection solution group in Table 1 of Example 1 of the present invention after being stored at 25°C for 14 days and culturing at 30°C for 72 hours, the results of most formulation groups The number of viable bacteria decreased significantly, and group 2 decreased the most, but the number of viable bacteria remained at 2 x 10 ⁸ CFU/g. Group 4 had the smallest decrease, with the number of viable bacteria reaching 1.2 x 10 ⁹ CFU/g, and maintaining a survival rate of more than 87% (not shown in the figure). From the culture results in Figure 13 after storage at 54°C for 14 days, it can be seen that the number of viable bacteria has dropped significantly. The number of viable bacteria in many groups has dropped below 10 ⁷ CFU/g, and only groups 3, 4 and 7 are still maintained. Above 10 ⁷ CFU/g, group 7 had the highest number of viable bacteria, reaching 6.2 x 10 ⁷ CFU/g. The freeze-dried mushroom of the present invention has good stability. It can maintain ^{a viable number of more than 2 x 10 8} CFU/g when stored at 25°C for 14 days, which is only about one lower than the freshly prepared freeze-dried mushroom. Log value.

without

Fig. 1 shows the number of viable bacteria of Pseudomonas fluorescens bacteria prepared with each protection solution group in Table 1 of Example 1 of the present invention after culturing at 30°C for 72 hours. Figures 2 to 4 respectively show the number of viable cells of yeasts 21849, 20405, and 22745 prepared with the protective liquid groups in Table 1 of Example 1 of the present invention after culturing at 25°C for 72 hours. Fig. 5 shows the number of viable bacteria of Pseudomonas fluorescens bacteria prepared in each protective solution group in Table 2 and Table 3 of Comparative Example 1 after culturing at 30°C for 72 hours. Fig. 6 shows the number of viable cells of yeasts 21849, 20405, and 22745 prepared by the protective liquid groups in Table 2 and Table 3 of Comparative Example 1 after culturing at 25°C for 72 hours. Fig. 7 shows the survival rate of Pseudomonas fluorescens bacteria prepared with each protection solution group in Table 1 of Example 1 of the present invention after culturing at 30°C for 72 hours. Figures 8 to 10 respectively show the survival rates of yeasts 21849, 20405, and 22745 prepared with the protective liquid groups in Table 1 of Example 1 of the present invention after culturing at 25°C for 72 hours. Fig. 11 shows the survival rate of Pseudomonas fluorescens bacterium prepared with each protection solution group in Table 2 and Table 3 of Comparative Example 1 after being cultured at 30°C for 72 hours. Fig. 12 shows the survival rates of yeasts 21849, 20405, and 22745 prepared by the protective liquid groups in Table 2 and Table 3 of Comparative Example 1 after culturing at 25°C for 72 hours. Figure 13 shows the Pseudomonas fluorescens bacterium prepared with the protection solution groups in Table 1 of Example 1 of the present invention and the results after storage at 25°C and 54°C for 14 days and culturing at 30°C for 72 hours Number of viable bacteria.

without

Claims (5)

A formula of bacterial protection agent, comprising: 1.125 to 4.5 parts by weight of trehalose; 2.25 to 4.5 parts by weight of skimmed milk powder; 1.125 to 2.25 parts by weight of mannitol; 0.0225 to 0.1125 parts by weight of vitamin C; 0.225 to 1.125 parts by weight Sodium glutamate; and 0.225 to 1.125 parts by weight of glycerin, wherein the bacterial protectant formula further contains Pseudomonas putida. For example, the bacterial protection agent formula of claim 1, which comprises the Pseudomonas putida and a group selected from the group consisting of the following groups 1 to 8: group 1: 1.125 parts by weight Trehalose; 2.25 parts by weight of skimmed milk powder; 1.125 parts by weight of mannitol; 0.0225 parts by weight of vitamin C; 0.225 parts by weight of sodium glutamate; and 0.225 parts by weight of glycerin; Group 2: 1.125 parts by weight of trehalose ; 2.25 parts by weight of skimmed milk powder; 2.25 parts by weight of mannitol; 0.1125 parts by weight of vitamin C; 1.125 parts by weight of sodium glutamate; and 1.125 parts by weight of glycerin; Group 3: 4.5 parts by weight of trehalose; 4.5 parts by weight of skimmed milk powder; 1.125 parts by weight of mannitol; 0.0225 parts by weight Parts of vitamin C; 1.125 parts by weight of sodium glutamate; and 1.125 parts by weight of glycerin; Group 4: 4.5 parts by weight of trehalose; 4.5 parts by weight of skimmed milk powder; 2.25 parts by weight of mannitol; 0.1125 parts by weight Vitamin C; 0.225 parts by weight of sodium glutamate; and 0.225 parts by weight of glycerin; Group 5: 1.125 parts by weight of trehalose; 4.5 parts by weight of skimmed milk powder; 1.125 parts by weight of mannitol; 0.1125 parts by weight of vitamin C ； 0.225 parts by weight of sodium glutamate; and 1.125 parts by weight of glycerin; Group 6: 1.125 parts by weight of trehalose; 4.5 parts by weight of skimmed milk powder; 2.25 parts by weight of mannitol; 0.0225 parts by weight of vitamin C; 1.125 parts by weight Parts of sodium glutamate; and 0.225 parts by weight of glycerin; Group 7: 4.5 parts by weight of trehalose; 2.25 parts by weight of skimmed milk powder; 1.125 parts by weight of mannitol; 0.1125 parts by weight of vitamin C; 1.125 parts by weight Sodium glutamate; and 0.225 parts by weight of glycerin; Group 8: 4.5 parts by weight of trehalose; 2.25 parts by weight of skimmed milk powder; 2.25 parts by weight of mannitol; 0.0225 parts by weight of vitamin C; 0.225 parts by weight of glutamine Sodium; and 1.125 parts by weight of glycerin. Such as claim 1 or 2 of the bacterial protection agent formulation, which is in powder form. For example, the bacterial protection agent formula of claim 1, which is composed of the following ingredients: 1.125 to 4.5 parts by weight of trehalose; 2.25 to 4.5 parts by weight of skimmed milk powder; 1.125 to 2.25 parts by weight of mannitol; 0.0225 to 0.1125 parts by weight 0.225 to 1.125 parts by weight of sodium glutamate; 0.225 to 1.125 parts by weight of glycerin; the inevitable impurities contained in the above components after purification; and Pseudomonas putida. A use for the cultivation of Pseudomonas putida (Pseudomonas putida) with a bacterial protective agent formula, the bacterial protective agent formula comprising: 1.125 to 4.5 parts by weight of trehalose; 2.25 to 4.5 parts by weight of skimmed milk powder; 1.125 to 2.25 parts by weight of mannitol; 0.0225 to 0.1125 parts by weight of vitamin C; 0.225 to 1.125 parts by weight of sodium glutamate; and 0.225 to 1.125 parts by weight of glycerin.

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