CN110897005A

CN110897005A - Method for sterilizing gram-negative pathogenic bacteria in milk

Info

Publication number: CN110897005A
Application number: CN201911123967.8A
Authority: CN
Inventors: 肖性龙; 曹怡芳; 余以刚
Original assignee: South China University of Technology SCUT
Current assignee: South China University of Technology SCUT
Priority date: 2019-11-15
Filing date: 2019-11-15
Publication date: 2020-03-24
Anticipated expiration: 2039-11-15
Also published as: CN110897005B

Abstract

The invention relates to a food sterilization technology, and particularly discloses a method for sterilizing gram-negative pathogenic bacteria in milk, which comprises the following steps: adding carvacrol and citral into milk, heating the milk at 40-55 deg.C until pathogenic bacteria are completely killed, and stopping heating to obtain sterile milk. According to the invention, the plant source compound is combined with mild heating treatment, and sterilization is performed cooperatively, so that an ideal sterilization effect on common gram-negative pathogenic bacteria in milk can be achieved; and has lower temperature and shorter sterilization time than pasteurization treatment. Meanwhile, the original nutritional value and flavor of the milk can be maintained to the maximum extent.

Description

Method for sterilizing gram-negative pathogenic bacteria in milk

Technical Field

The invention relates to a sterilization technology in food, in particular to a sterilization method of gram-negative pathogenic bacteria in milk.

Background

Milk is one of the oldest natural beverages and is rich in proteins, fats, vitamins and minerals. The milk is rich in nutrition, easy to digest and absorb, high in quality, low in price and convenient to eat, and is a main food of human daily diet. However, milk is also susceptible to the growth of various microorganisms, particularly some gram-negative pathogenic bacteria: escherichia coli, Salmonella, Cronobacter sakazakii, and the like. Therefore, it is very important to control the pathogenic bacteria in milk. Is commonly used for sterilizing milkThe method is a traditional pasteurization method and mainly comprises three steps: low temperature long time sterilization (63 deg.C, 30min), high temperature short time sterilization (72 deg.C, 15s), and ultra high temperature instantaneous sterilization (138 deg.C, 2-4 s). The sterilization methods can kill most harmful pathogenic bacteria in the milk, but can cause adverse effects to different degrees on nutrient substances, texture and flavor in the milk; and the equipment required for pasteurization is expensive, and needs to be cleaned and monitored in time, so that the cost is high. In recent years, non-thermal sterilization techniques have been used for inactivation of food-borne pathogenic bacteria in milk, such as high-voltage pulsed electric field, ultra-high voltage, ultrasonic waves and SC-CO₂The technologies can achieve good antibacterial effect under the optimal conditions, but the sub-lethal damage to cells is caused after the treatment of the ultrahigh-voltage and high-voltage pulse electric field, and potential harm is caused in the subsequent storage process of the milk; the sterilization effect of the ultrasonic wave is influenced by more factors, and whether the ultrasonic wave can be applied to the dairy industry needs further verification; SC-CO₂The equipment is expensive and the operation is complex, so the popularization in the dairy industry is difficult.

Recently, there has been a trend towards the use of plant-derived compounds for bacteriostasis, which are considered to be non-toxic and harmless, and many of which have been approved for use as food additives. However, when the plant source compound is used alone, the ideal bacteriostatic effect can be achieved only at a high bacteriostatic concentration, so that the food flavor is also adversely affected.

Disclosure of Invention

The invention aims to provide a method for sterilizing gram-negative pathogenic bacteria in milk. The method of the invention applies the plant source compound with synergistic bactericidal effect, and then is assisted with medium-low temperature heating treatment for a period of time, so that all bacteria in the milk can be killed, and the original nutritional quality and flavor of the milk can be maintained. The invention can replace or partially replace pasteurization more effectively.

The object of the invention is achieved by the following steps:

a method for killing gram-negative pathogenic bacteria in milk comprises the following steps:

adding carvacrol and citral into milk, heating the milk at 40-55 deg.C until pathogenic bacteria are completely killed, and stopping heating to obtain sterile milk; when the heating temperature is more than or equal to 40 and T is less than 45 ℃, the concentration of carvacrol in the milk is more than or equal to 200 mu g/mL, and the concentration of citral is more than or equal to 1600 mu g/mL; when the heating temperature is more than or equal to 45 ℃ and T is less than 50 ℃, the concentration of carvacrol in the milk is more than or equal to 100 mu g/mL, and the concentration of citral is more than or equal to 800 mu g/mL; when the heating temperature T is not less than 50 ℃ and not more than 55 ℃, the concentration of carvacrol in the milk is not less than 50 mug/mL, and the concentration of citral is not less than 400 mug/mL.

Preferably, when the heating temperature T is more than or equal to 45 ℃ and less than 50 ℃, the concentration of carvacrol in the milk is more than or equal to 200 mu g/mL, and the concentration of citral is more than or equal to 1600 mu g/mL; when the heating temperature T is not less than 50 ℃ and not more than 55 ℃, the concentration of carvacrol in the milk is not less than 100 mu g/mL, and the concentration of citral is not less than 800 mu g/mL.

Preferably, the heating time is 5-90 min.

Preferably, the heating time is 10-30 min.

Preferably, the heating temperature is more than or equal to 50 and less than or equal to 55 ℃, and the heating time is 15 +/-5 min.

The concentration of the plant source compound is the final concentration of the plant source compound added into a milk system.

Compared with the existing pasteurization technology, the invention has the following advantages and technical effects:

(1) according to the invention, the plant source compound is combined with mild heating treatment, so that a synergistic sterilization effect can be achieved; and has lower temperature and shorter sterilization time than pasteurization treatment.

(2) Simple use, low cost and high cost-effectiveness ratio.

(3) The plant source compound is natural, nontoxic, and edible, and can be used as food additive.

(4) The ideal sterilization effect is achieved, and meanwhile, the original nutrition, texture and flavor of the milk can be kept to the maximum extent.

Drawings

FIG. 1 shows the bacteriostatic effect of carvacrol and citral at different concentrations in example 3 on E.coli in milk at different heating temperatures, A, B, C, D representing heating temperatures of 40, 45, 50 and 55 deg.C, respectively.

Fig. 2 is the bacteriostatic effect of carvacrol and citral at different concentrations in example 4 on salmonella in milk at different heating temperatures, A, B, C, D representing heating temperatures of 40, 45, 50 and 55 ℃ respectively.

Fig. 3 is the bacteriostatic effect of carvacrol and citral at different concentrations in example 5 on cronobacter sakazakii in milk at different heating temperatures, A, B, C, D representing heating temperatures of 40, 45, 50 and 55 ℃ respectively.

FIG. 4 shows the inactivation effect of the optimal sterilization conditions on the mixed bacteria liquid of different gram-negative pathogenic bacteria in milk in example 6 of the present invention.

Detailed Description

To further describe the present invention, the present invention will be described in further detail with reference to examples. The reagents and methods used in the present invention are, unless otherwise specified, those commonly used in the art and conventional methods.

Example 1

Screening the plant source compound with good antibacterial effect:

selecting ten plant source compounds: carvacrol, thymol, eugenol, citral, protocatechualdehyde, caprylic acid, gallic acid, ferulic acid, resveratrol and allicin. Determination of different botanical compounds on common gram-negative bacilli: minimum Inhibitory Concentrations (MIC) of escherichia coli, salmonella, and cronobacter sakazakii. A mother liquor of ten plant source compounds was prepared using 50% aqueous ethanol. Adding 100 μ L sterile TSB culture medium into each well of 96-well plate, adding 100 μ L carvacrol mother liquor into each well, diluting with equal times, mixing well, adding 100 μ L carvacrol mother liquor with 10 concentration into each well⁶And repeatedly pumping the bacterial suspension with CFU/mL to ensure that the final concentration of carvacrol is 6.4, 4.8, 3.2, 2.4, 1.6, 1.2, 0.8, 0.6, 0.4, 0.2, 0.1 and 0.05mg/mL in sequence. The samples were then incubated in an incubator at 37 ℃ for 24 h. Similarly, the minimum inhibitory concentrations of the other nine compounds against different bacteria were determined according to the above procedure, and the results are detailed in table 1. The experiment was carried out with a medium containing 0.1mg/mL ampicillinAs a positive control, TSB without the plant source compound was a negative control. MIC was defined as the lowest concentration of the plant-derived compound that inhibited growth of e.coli, salmonella and cronobacter sakazakii, respectively, under macroscopic observation.

Example 2

Screening combinations of plant source compounds with synergistic effects according to the invention:

according to the results of example 1, 4 plant source compounds with better bacteriostasis effect are selected: carvacrol, citral, thymol and caprylic acid to determine the fractional inhibitory concentration coefficient of different compounds when used in combination. Mother liquors with carvacrol, citral, caprylic acid and thymol concentrations of 102.4mg/mL were prepared using 50% aqueous ethanol. And diluted with TSB medium to give solutions of each compound at concentrations of 4 × MIC, 2 × MIC, 1 × MIC, 1/2, 1/4, 1/8MIC, respectively. Preparing different bacteria with concentration of 10⁶CFU/mL suspension, 100. mu.L of the above-mentioned suspension was added to each well of a 96-well plate. 50 mu L of carvacrol and citral with different concentrations are respectively added into the horizontal and vertical wells of a 96-well plate to obtain the final concentrations (2 × MIC, 1 × MIC, 1/2, 1/4, 1/8 and 1/16MIC), the final concentrations are repeatedly blown and beaten after the addition, the mixture is evenly mixed, and the mixture is placed at 37 ℃ for culturing for 24 hours. Samples that inhibited the growth of cronobacter sakazakii were labeled, and FIC values corresponding to different compounds in each sample were calculated. Three replicates were made for each sample. And other five combinations: carvacrol and caprylic acid, carvacrol and thymol, citral and caprylic acid, citral and thymol, caprylic acid and thymol FIC value determination the procedure was followed for carvacrol and citral.

FICI (FICI) refers to the combination of Fractional Inhibitory concentrations of each Inhibitory compound (A or B), and is calculated as follows:

FICI interpretation criteria: when the FICI is less than or equal to 0.5, the two antibacterial compounds have synergistic action; when the FICI is more than 0.5 and less than or equal to 1, the two bacteriostatic compounds have additive action; when the FICI is more than 1 and less than or equal to 4, the two antibacterial compounds have irrelevant effects; when FICI > 4, the two bacteriostatic compounds are antagonistic.

Example 3

Inactivation effect of combined temperature of two plant source compounds on escherichia coli in milk

According to the results of example 2, a combination of carvacrol and citral with synergistic effects was selected for the subsequent examples. Adding commercially available milk 10mL into sterilized test tube, heating in 65 deg.C water bath for 30min for pasteurization, cooling to room temperature, adding 100 μ L of prepared 10% concentration milk⁸CFU/mL of E.coli suspension, appropriate amounts of carvacrol and citral were added to the test tube to concentrations of (0+0, 1/3MIC +1/3MIC, 1/2MIC +1/2MIC, 2/3MIC +2/3MIC, MIC + MIC, 2MIC +2MIC), respectively, and no plant source compound was added to the sample control group. The inoculated samples are placed at 4 different temperatures (40, 45, 50 and 55 ℃) for heat preservation and sterilization. Samples at 40 ℃ and 45 ℃ are respectively sterilized at a heat preservation time of 0, 20, 40, 60, 80, 100 and 120min, samples at 50 ℃ and 55 ℃ are respectively sterilized at a heat preservation time of 0, 5, 10, 20, 40, 60 and 90min, the samples are directly diluted or diluted by 10 times by PBS, the diluted samples are coated on a TSA culture medium to be cultured for 24 hours at a constant temperature of 37 ℃, the total number of bacteria is recorded, and 3 parallels are set for each group of experiments.

Figure 1 shows that the combination temperature treatment of carvacrol and citral can inhibit the growth of escherichia coli more obviously than the combination or temperature single action of carvacrol and citral, and the inhibition effect is more obvious when the temperature is higher. The combination of carvacrol and citral 1/3MIC, 1/2MIC, 2/3MIC, MIC and 2MIC can completely kill Escherichia coli in milk after being acted at 55 deg.C, 50 deg.C and 50 deg.C for 90, 60, 40, 20 and 10min respectively.

Example 4

Inactivation effect of two plant source compounds on salmonella in milk by combined temperature

Adding commercially available milk 10mL into sterilized test tube, heating in 65 deg.C water bath for 30min for pasteurization, cooling to room temperature, adding 100 μ L of prepared 10% concentration milk⁸CFU/mL of Salmonella suspension, adding appropriate amount of carvacrol and citral into test tube to give concentration of (0+0, 1/3MIC + 1/3)MIC, 1/2MIC +1/2MIC, 2/3MIC +2/3MIC, MIC + MIC, 2MIC +2MIC), sample control group without added botanical compound. The inoculated samples are placed at 4 different temperatures (40, 45, 50 and 55 ℃) for heat preservation and sterilization. Samples at 40 ℃ and 45 ℃ are respectively sterilized at the temperature of 0, 20, 40, 60, 80, 100 and 120min in a heat preservation way, samples at 50 ℃ and 55 ℃ are respectively sterilized at the temperature of 0, 5, 10, 20, 40, 60 and 90min in a heat preservation way, the samples are directly diluted or diluted by 10 times by PBS, the diluted samples are coated on a TSA culture medium to be cultured for 24h at the constant temperature of 37 ℃, the total number of bacteria is recorded, and 3 parallels are set for each group of experiments.

Fig. 2 shows that the combination of carvacrol and citral with temperature treatment can inhibit the growth of salmonella more significantly than the combination of carvacrol and citral or the effect of temperature alone, and the higher the temperature, the more significant the inhibition effect. The combination of carvacrol and citral 1/3MIC, 1/2MIC, 2/3MIC, MIC and 2MIC can completely kill salmonella in milk after 90min, 60 min, 40 min, 20min and 10min respectively at 55 ℃, 50 ℃ and 50 ℃.

Example 5

Inactivation effect of two plant source compounds on cronobacter sakazakii in milk by combining temperature

Adding commercially available milk 10mL into sterilized test tube, heating in 65 deg.C water bath for 30min for pasteurization, cooling to room temperature, adding 100 μ L of prepared 10% concentration milk⁸CFU/mL of a suspension of cronobacter sakazakii, appropriate amounts of carvacrol and citral were added to the test tube to concentrations of (0+0, 1/3MIC +1/3MIC, 1/2MIC +1/2MIC, 2/3MIC +2/3MIC, MIC + MIC, 2MIC +2MIC), respectively, and the sample control group was not added with the plant source compound. The inoculated samples are placed at 4 different temperatures (40, 45, 50 and 55 ℃) for heat preservation and sterilization. Samples at 40 ℃ and 45 ℃ are respectively sterilized at a heat preservation time of 0, 20, 40, 60, 80, 100 and 120min, samples at 50 ℃ and 55 ℃ are respectively sterilized at a heat preservation time of 0, 5, 10, 20, 40, 60 and 90min, the samples are directly diluted or diluted by 10 times by PBS, the diluted samples are coated on a TSA culture medium to be cultured for 24 hours at a constant temperature of 37 ℃, the total number of bacteria is recorded, and 3 parallels are set for each group of experiments.

Fig. 3 shows that the combination temperature treatment of carvacrol and citral can inhibit the growth of cronobacter sakazakii more obviously compared with the combination of carvacrol and citral or the single action of temperature, and the higher the temperature is, the more obvious the inhibition effect is. The combination of carvacrol and citral 1/3MIC, 1/2MIC, 2/3MIC, MIC and 2MIC can completely kill Cronobacter sakazakii in milk after acting at 55 ℃, 50 ℃ and 50 ℃ for 90, 60, 40, 20 and 10min respectively.

Example 6

According to the results of examples 3, 4 and 5, optimal conditions are selected, and the survival of three gram-negative pathogenic bacteria in the milk under the optimal conditions is determined. Adding commercially available milk 10mL into sterilized test tube, heating in 65 deg.C water bath for 30min for pasteurization, cooling to room temperature, adding 100 μ L of prepared 10% concentration milk⁸CFU/mL of mixed bacterial suspension of Escherichia coli, Salmonella and Cronobacter sakazakii, appropriate amounts of carvacrol and citral (concentration: MIC + MIC) were added to the test tube, and no plant source compound was added to the control group. And (3) placing the inoculated milk at 50 ℃ for heat preservation and sterilization, respectively preserving the heat for 0, 5, 10, 15, 10, 25 and 30min, directly diluting the sample or diluting the sample by 10 times by using PBS, coating the diluted sample on a TSA culture medium, culturing for 24h at the constant temperature of 37 ℃, recording the total number of bacteria, and setting 3 parallels in each group of experiments.

FIG. 4 shows that under the selected optimal treatment condition, the MIC combination of carvacrol and citral is subjected to warm treatment at 50 ℃, and the mixed bacteria liquid of 6Log CFU/mL escherichia coli, salmonella and cronobacter sakazakii in milk can be completely killed after 15 min.

Table 1 shows the minimum inhibitory concentrations of ten plant-derived compounds against different gram-negative bacilli

Table 1 shows that the MICs of resveratrol and allicin in ten selected substances are all higher than 5mg/mL, and the bacteriostatic effect is the worst; the carvacrol and the thymol have the best antibacterial effect, and in aldehydes, citral has a better antibacterial effect; among acids, caprylic acid has a good bacteriostatic effect.

Table 2 shows that of the six combinations of four botanical source compounds, only the combination of carvacrol and citral showed synergistic effects on all three pathogens.

Claims

1. A method for sterilizing gram-negative pathogenic bacteria in milk is characterized by comprising the following steps:

2. The sterilization method as claimed in claim 1, wherein when the heating temperature T is more than or equal to 45 ℃ and less than 50 ℃, the concentration of carvacrol in the milk is more than or equal to 200 μ g/mL, and the concentration of citral is more than or equal to 1600 μ g/mL; when the heating temperature T is not less than 50 ℃ and not more than 55 ℃, the concentration of carvacrol in the milk is not less than 100 mu g/mL, and the concentration of citral is not less than 800 mu g/mL.

3. The sterilization method according to claim 1 or 2, wherein the heating time is 5 to 90 min.

4. The sterilization method according to claim 3, wherein the heating time is 10 to 30 min.

5. The sterilization method according to claim 4, wherein the heating temperature T is 50 ℃ or more and 55 ℃ or less, and the heating time is 15 +/-5 min.