CN113355254B - Screwdriver protective agent formula and its use - Google Patents

Abstract

The invention relates to a starter protector formula and application thereof. A starter protectant formulation comprising: 1.125+ -10% to 4.5+ -10% trehalose by weight; 2.25+ -10% to 4.5+ -10% of skimmed milk powder; mannitol in an amount of 1.125.+ -. 10% to 2.25.+ -. 10%; 0.0225 + -10% to 0.1125 + -10% by weight of vitamin C;0.225 + -10% to 1.125 + -10% by weight of sodium glutamate; and 0.225.+ -. 10% to 1.125.+ -. 10% by weight of glycerol.

Description

Screwdriver protective agent formula and its use

Technical Field

The invention relates to a starter protective agent formula, in particular to a starter protective agent formula which is applicable to both gram-positive bacteria and gram-negative bacteria.

Background technique

Since the number of natural microorganisms is usually not enough for actual use, and it is also very difficult to preserve them. They are often stimulated by the environment or mutated after multiple generations of cultivation. Therefore, they are mostly used and circulated in the form of "starters" that have been treated with hot air drying or freeze drying. Taking the food industry as an example, due to its large operating volume, if microorganisms are required for fermentation in the process, natural bacteria cannot be used directly. Domesticated starters are often used for pre-culture expansion, and subsequent processes are carried out only after a sufficient number of bacteria is reached. The actual industrial application range includes industries that require yeast as raw materials, such as beer fermentation and bread baking industries. In addition, in the biological preparation industry used in agriculture, the biological preparation products produced also face the requirement of high and stable viable bacterial counts, so they are also suitable for pre-culture expansion with starters. Therefore, after discovering the characteristic potential strains, a starter formula is needed that can achieve the purpose of high stability and high viable bacterial counts, so as to increase the application flexibility in the process. At the same time, the bacteria can also be sold in the form of starters to increase 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 comprises skimmed milk powder, mannitol, sodium glutamate or glycine, corn starch and trehalose.

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

The current screwdriver protection agent formula has the disadvantage of low live bacterial count during cultivation, especially when the screwdriver protection agent formula is used after a period of time after preparation, the live bacterial count drops significantly.

Summary of the invention

The main purpose of the present invention is to provide a screwdriver protection agent formula, which can improve the shortcomings of low viable bacterial count and survival rate, especially a screwdriver protection agent formula with improved stability, which still has a comparable viable bacterial count after storage for a period of time compared with the screwdriver protection agent formula just prepared.

A screwdriver protective agent formulation completed according to the purpose of the present invention comprises:

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 glycerol.

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

The screwdriver protective agent formula of the present invention has a characteristic that it has improved viable bacteria count and survival rate no matter it is used for culturing Gram-positive bacteria or Gram-negative bacteria.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 shows the number of live bacteria of Pseudomonas putida starters prepared with the protective solution groups in Table 1 of Example 1 of the present invention after being cultured at 30° C. for 72 hours.

2 to 4 respectively show the number of viable bacteria of yeast starters 21849, 20405, and 22745 prepared with the protective solution groups in Table 1 of Example 1 of the present invention after being cultured at 25° C. for 72 hours.

FIG. 5 shows the number of live bacteria of Pseudomonas putida starters prepared with each protective solution group in Table 2 and Table 3 of Control Example 1 after being cultured at 30° C. for 72 hours.

FIG6 shows the number of viable yeast starters 21849, 20405, and 22745 prepared with the protective solution groups in Table 2 and Table 3 of Control Example 1 after being cultured at 25° C. for 72 hours.

FIG. 7 shows the survival rate of Pseudomonas putida starters prepared with each protective solution group in Table 1 of Example 1 of the present invention after being cultured at 30° C. for 72 hours.

8 to 10 respectively show the survival rates of yeast starters 21849, 20405, and 22745 prepared with the various protective solution groups in Table 1 of Example 1 of the present invention after being cultured at 25° C. for 72 hours.

FIG. 11 shows the survival rate of Pseudomonas putida starters prepared with each protective solution group in Table 2 and Table 3 of Control Example 1 after being cultured at 30° C. for 72 hours.

FIG. 12 shows the survival rates of yeast starters 21849, 20405, and 22745 prepared with the protection solution groups in Table 2 and Table 3 of Control Example 1 after being cultured at 25° C. for 72 hours.

13 shows the viable bacterial counts of Pseudomonas putida starters prepared with the protective solution groups in Table 1 of Example 1 of the present invention and after being stored at 25° C. and 54° C. for 14 days and then cultured at 30° C. for 72 hours.

Detailed ways

The present invention and its improvements will be further understood by the following examples and comparative examples. The following materials and instruments are used in the following examples and comparative examples.

Material

Skim milk powder MERCK Casein MERCK glycerin SIGMA Vitamin C SIGMA Trehalose SIGMA sucrose SIGMA Monosodium glutamate (MSG) Weiquan Span 60 SIGMA YM Broth Gibico

Pseu F agar medium recipe

Tryptone 1% MERCK Tryptic protein 1% MERCK Potassium hydrogen phosphate (K ₂ HPO ₄ ) 0.15% SIGMA Magnesium sulfate (MgSO ₄ ) 0.15% SIGMA Agar SIGMA

instrument

Pseudomonas putida culture method

Pseudomonas putida YLW01 was selected as the experimental strain. YLW01 was cultured in a medium containing 1% Molasses, 1% MSG, 1.5 g/L K ₂ HPO ₄ and 1.5 g/L MgSO ₄ ^. 7H ₂ O (50 mL medium/250 mL Erlenmeyer flask) at 30°C and 150 rpm for 42 hr. After the culture was completed, the culture was centrifuged at 8000 rpm for 10 minutes at 4°C, the supernatant was discarded, and the culture was washed with water and centrifuged again, and this washing method was repeated twice. The centrifuged bacterial sludge was then taken out and stored in a refrigerator.

Yeast culture method

Yeast strains 21849, 20405, and 22745 were selected as experimental strains. Yeast strains 21849, 20405, and 22745 were cultured in YMbroth (Becton Dickinson and Company) medium (50 mL medium/250 mL Erlenmeyer flask) at 25°C and 250 rpm for 48 hr. After the culture was completed, the culture was centrifuged at 8000 rpm for 10 minutes at 4°C, the supernatant was discarded, and the culture was washed with water and centrifuged again, and this washing method was repeated twice. The centrifuged bacterial sludge was then taken out and stored in a refrigerator.

Preservative formula/preparation of protective solution

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 evenly dissolved. The pH is then adjusted to 7 or 8 with the following phosphate solution (adjusted from alkaline to acidic) or glycine-sodium hydroxide buffer (adjusted from acidic to alkaline).

Table 1: Protective agent formula (g) / protective solution (g/vol %)

Glycine-sodium hydroxide buffer preparation:

50 mL of glycine aqueous solution (0.2 M) and 8.8 mL of sodium hydroxide aqueous solution (0.2 M) were mixed evenly and then quantified to 200 mL.

Preparation of phosphate solution:

87.7 mL of sodium dihydrogen phosphate aqueous solution (0.2 M) and 12.3 mL of sodium monohydrogen phosphate aqueous solution (0.2 M) were mixed evenly and then quantified to 200 mL.

Example 1

Preparation of Screwdriver

The prepared bacterial sludge was taken out from the refrigerator, and 7.5 g of the bacterial sludge was mixed with each protective solution in Table 1, respectively, and stirred for 30 minutes until the mixture was uniform. The mixture was pre-frozen at -20°C and freeze-dried to obtain a starter.

Take 1 g of the freshly prepared freeze-dried starter and dissolve it back into 10 mL of water, and make a series of dilutions with appropriate multiples. Take 100 μL of the starter dilution solution and spread it on Pseu F medium. Incubate it at 30°C for 72 hours and then count the colonies.

Comparative Example 1

The opener was prepared according to the formula of CN106244459 and CN107828683. 22.5 mL of the protective solution in Table 2 and Table 3 were mixed with 7.5 g of the prepared bacterial mud, stirred for 30 minutes until the mixture was uniform, and then pre-frozen at -20°C and freeze-dried to obtain the opener of Control Example 1.

Take 1 g of the freshly prepared freeze-dried starter and dissolve it in 10 mL of water. Then make a series of dilutions with appropriate multiples. Apply 100 μL of the starter dilutions on Pseu F medium and incubate at 30°C for 72 hours before counting the colonies.

Table 2 CN106244459 protective solution (g/vol %)

Group Trehalose(%) Skim 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 protective solution (g/vol %)

Group Trehalose(%) Skim milk powder(%) glycerin(%) Mannitol(%) sucrose(%) ascorbic acid water CN107828683 6 8 1 2 4 0.5 78.5

result

FIG1 shows the viable count of Pseudomonas putida starters prepared with each protective solution group in Table 1 of Example 1 after culturing at 30°C for 72 hours. As shown in FIG1 , the viable count of Pseudomonas putida was maintained above 10 ⁹ CFU/g, indicating that the protective agent of the present invention has a good protective effect. Among them, Group 2 has the highest viable count of 6.3x10 ⁹ CFU/g.

Figures 2 to 4 show the viable counts of yeasts 21849, 20405, and 22745 starters prepared with the protective solution groups in Table 1 of Example 1 after being cultured at 25°C for 72 hours. The highest viable counts of yeasts 21849, 20405, and 22745 were 3.44x10 ⁹ CFU/g (Group 5 in Figure 2), 3.75x10 ⁹ CFU/g (Group 7 in Figure 3), and 5x10 ⁹ CFU/g (Group 3 in Figure 4), respectively.

Figure 5 shows the viable bacterial counts of Pseudomonas putida starters prepared with the protective solution groups in Table 2 and Table 3 of Control Example 1 after culturing at 30°C for 72 hours. As shown in Figure 5 , the viable bacterial counts of Pseudomonas putida are 5.8x10 ⁷ , 8.8x10 ⁸ and 2.6x10 ⁸ CFU/g, respectively.

Figure 6 shows the viable counts of yeast 21849, 20405, and 22745 starters prepared with the protective solution groups in Table 2 and Table 3 of Control Example 1 after culturing at 25°C for 72 hours. The culturing effects were all poor, with most viable counts at 10 ⁸ CFU/g. The group with the highest viable count was yeast 22745 using the formula CN106244459-2, reaching 1.99x10 ⁹ CFU/g.

FIG7 shows the survival rate of Pseudomonas putida starters prepared with each protective solution group in Table 1 of Example 1 after being cultured at 30° C. for 72 hours. The survival rate calculation method is as follows:

Survival rate (%) = [number of viable bacteria after freeze-drying (CFU) / number of bacteria added before freeze-drying (CFU)] * 100%

It can be seen in Figure 7 that the viable counts of Pseudomonas putida were all maintained above 15%, with Group 2 having the highest survival rate of about 78%. The performance of the survival rates of the groups in Figure 7 is similar to the performance of the viable counts in Figure 1.

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

Figure 11 shows the survival rates of Pseudomonas putida starters prepared with the protective solution groups in Table 2 and Table 3 of Control Example 1 after being cultured at 30°C for 72 hours. As shown in Figure 11, the survival rates of Pseudomonas putida were 0.07%, 1.02% and 0.3%, respectively.

FIG12 shows the survival rate of yeast 21849, 20405, and 22745 starters prepared with each protective solution group in Table 2 and Table 3 of Control Example 1 after culturing at 25° C. for 72 hours. The survival rate shown in FIG12 has the same trend as the number of viable cells in FIG6 , which is about 2%, and the highest group is the culture of CN106244459-1 in yeast 20405.

As can be seen from Figures 1 to 12 above, the formula of the present invention and the previous formula were tested with Pseudomonas putida and three yeasts (21849, 20405, 22745), and it was found that the number of live bacteria and the survival rate of the formula of the present invention were significantly improved compared with the previous formulas CN106244459-1, CN106244459-2 and CN107828683. This proves that the present invention has unexpected enhancement effect.

Example 2: Freeze-dried starter stability test

The freeze-dried starter prepared in Example 1 was stored at 25°C and 54°C for 14 days, 1 g of the starter was dissolved in 10 mL of water, and a series of dilutions of appropriate multiples were performed. 100 μL of the Pseudomonas putida starter dilutions were smeared on PseuF medium, and the culture was placed at 30°C for 72 hours, and then the colonies were counted.

The results are shown in FIG13 , which also shows the viable count of the freshly prepared freeze-dried starter. FIG13 shows that after the Pseudomonas putida starter prepared with each protective solution group in Table 1 of Example 1 of the present invention was stored at 25°C for 14 days and then cultured at 30°C for 72 hours, the viable count of most formula groups decreased significantly, and the decrease in group 2 was the largest, but the viable count was still maintained at 2 x 10 ⁸ CFU/g. Group 4 had the smallest decrease, with the viable count reaching 1.2 x 10 ⁹ CFU/g, and maintained a survival rate of more than 87% (not shown in the figure). From the culture results after storage at 54°C for 14 days in FIG13 , it can be found that the viable count has dropped significantly, and the viable count of most groups has dropped to below 10 ⁷ CFU/g, and only groups 3, 4 and 7 still maintain above 10 ⁷ CFU/g, and the viable count of group 7 is the highest, reaching 6.2 x 10 ⁷ CFU/g. The freeze-dried starter of the present invention has good stability and can maintain a viable bacterial count of more than 2 x 10 ⁸ CFU/g after being stored at 25° C. for 14 days, which is only about one logarithm lower than that of the freshly prepared freeze-dried starter.

Claims (8)

1. A screwdriver protective agent formulation, which consists of the following ingredients: 1.125±10% to 4.5±10% by weight of trehalose; 2.25±10% to 4.5±10% by weight of skimmed 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 sodium glutamate; and 0.225±10% to 1.125±10% by weight of glycerol. 2. The screwdriver protecting agent formulation as claimed in claim 1, which is composed of the following ingredients: 1.125±5% to 4.5±5% by weight of trehalose; 2.25±5% to 4.5±5% by weight of skimmed milk powder; 1.125±5% to 2.25±5% by weight of mannitol; 0.0225±5% to 0.1125±5% by weight of vitamin C; 0.225±5% to 1.125±5% by weight of sodium glutamate; and 0.225±5% to 1.125±5% by weight of glycerol. 3. The screwdriver protective agent formulation as claimed in claim 2, which is selected from the following group: Group 1: 1.125 parts by weight of 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 glycerol; 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 glycerol; 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 of vitamin C; 1.125 parts by weight of sodium glutamate; and 1.125 parts by weight of glycerol; 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 of vitamin C; 0.225 parts by weight of sodium glutamate; and 0.225 parts by weight of glycerol; 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 glycerol; 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 of sodium glutamate; and 0.225 parts by weight of glycerol; 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 of sodium glutamate; and 0.225 parts by weight of glycerol; 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 sodium glutamate; and 1.125 parts by weight of glycerol. 4. The screwdriver protective agent formulation as claimed in claim 1, which is in powder form. 5. The screwdriver protective agent formulation as claimed in claim 1, which is composed of the following ingredients: 1.125±10% to 4.5±10% by weight of trehalose; 2.25±10% to 4.5±10% by weight of skimmed 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 sodium glutamate; 0.225±10% to 1.125±10% by weight of glycerol; and The inevitable impurities contained in the above ingredients after refining. 6. The screwdriver protecting agent formulation according to any one of claims 1 to 5, wherein the weight portion of the skimmed milk powder is less than 4.5 weight portions and the weight portion of the mannitol is less than 2.25 weight portions. 7. Use of the starter protecting agent formulation as claimed in claim 1 in the cultivation of yeast. 8. Use of the screwdriver protecting agent formulation as claimed in claim 1 in the cultivation of Pseudomonas putida.