CN121406627A - Preparation method of yogurt starter - Google Patents

Abstract

The invention relates to a preparation method of a yogurt starter. The preparation method comprises the steps of fermenting and culturing lactobacillus, detecting at least one of dielectric constant, viable count, fermentation activity or dry weight of thallus in fermentation process, judging fermentation end point, collecting thallus, mixing thallus with embedding agent to obtain mixed solution, granulating and drying the mixed solution to obtain the yogurt starter. The invention designs a preparation process of the yogurt starter, accurately collects lactic acid bacteria in a specific growth period through real-time online detection, designs a specific thallus embedding agent, efficiently protects the thallus, designs a specific liquid nitrogen granulation process, shortens the low-temperature freezing time of the thallus, can prepare uniform granular liquid drops through a liquid drop tray, directly performs sublimation drying, shortens the drying period, simultaneously can reduce the production cost, increases the solubility of the starter, and obtains the yogurt starter with good solubility, activity, fermentation activity and stability.

Description

Preparation method of yogurt starter

Technical Field

The invention belongs to the technical field of microorganisms, and relates to a preparation method of a yogurt starter.

Background

Starter cultures are a type of bacterial or microbial cultures with a high bacterial count, which have the ability to break down lactose to produce lactic acid and are often used for the production of fermented products such as cheese, yoghurt, cream, etc. According to the development process of the yoghurt starter and the manufacturing process thereof, the yoghurt starter can be divided into 3 types, namely a natural starter, a secondary starter and a direct starter. The product can keep unique taste and flavor through the fermentation of microorganisms, contains a large amount of active lactobacillus, can regulate the balance of intestinal flora, and improves the immune function of organisms.

At present, a large amount of direct-vat starter is used, and the direct-vat starter is directly added into a dry powder starter or a frozen concentrated starter in a fermentation tank without passage in the process of preparing the yoghurt, so that an intermediate subculture process is not needed, and the use is convenient. The characteristics of the starter (fermentation activity, post-acid, viable count, storage stability and the like) can directly influence the quality of the fermentation product, and the factors influencing the quality of the starter are mainly the production and preparation process except the difference among strains.

Along with the continuous improvement of the living standard of people, the quality requirements on the starter product are also higher and higher. The conventional direct-vat starter mainly comprises the steps of fermenting lactobacillus, centrifugally collecting thalli, adding a protective agent, performing vacuum freeze drying and crushing to obtain a powdery starter, and for example, CN117586913A discloses a preparation method and application of the direct-vat starter. However, the freeze-drying process has longer freeze-drying period and higher production cost, the viable count and the survival rate of the thalli can be affected to a certain extent due to long low-temperature pre-freezing time, so that the performance of the fermenting agent is affected, and in addition, the phenomenon of caking, suspension and uneven dispersion can sometimes occur in the aspect of solubility due to the fact that the fermenting agent is in a powder shape.

In view of the above, development of a method for preparing a high-activity, high-solubility and high-storage-stability starter is of great significance to the field of starter application.

Disclosure of Invention

Aiming at the defects and actual demands of the prior art, the invention provides a preparation method of a yogurt starter for preparing a starter with high activity, and has good solubility and stability, short drying period and low production cost.

In order to achieve the above purpose, the invention adopts the following technical scheme:

The invention provides a preparation method of a yogurt starter, which comprises the following steps:

Fermenting and culturing lactobacillus, detecting at least one of dielectric constant, viable count, fermentation activity or dry weight of thallus of fermentation liquid in fermentation process, judging fermentation end point, and collecting thallus;

mixing thalli with an embedding agent to obtain a mixed solution, and granulating and drying the mixed solution to obtain the yogurt starter;

The embedding agent contains trehalose, betaine, tertiary butanol, phospholipid, dibutyl phthalate, sodium polyacrylate, beta-cyclodextrin and hydroxypropyl starch.

The invention designs a preparation method of the yogurt starter, which accurately collects lactic acid bacteria growing in a specific period by characterizing fermentation activity and the like in real time, designs a specific embedding agent, efficiently protects thalli, and performs granulation and drying treatment on the obtained mixed liquid to obtain the starter with high activity, high solubility and stability, and has short drying period and low production cost.

In the invention, the dielectric constant (pF/cm) can be detected by adopting an online living cell detection electrode and the like, and can be linearly fitted with the fermentation activity to characterize the fermentation activity online in real time.

According to the invention, the fermentation end point can be judged according to the dielectric constant, the viable count, the fermentation activity or the dry weight of the bacteria and the like of the fermentation liquid, and the fermentation is stopped when each parameter reaches the target value (when the beginning of the reduction is detected), so that the collection of the lactic acid bacteria in a specific growth period is realized accurately.

The invention designs an embedding agent with specific composition, trehalose can provide corresponding hydrogen bonds when the thalli lose water in the drying process, ensures the normal structural stability of the thalli, betaine and tertiary butanol are used for maintaining the osmotic pressure balance inside and outside the thalli cells and play a role in the cells, lecithin can maintain the fluidity of the cell membranes of the thalli, keep the cell membranes of the thalli cells to be functionally complete in the drying process, further maintain the activity of the thalli, and two macromolecular substances of beta-cyclodextrin and hydroxypropyl starch can be wrapped on the surface of the thalli, and simultaneously dibutyl phthalate is added to enhance the wrapping tightness degree of the macromolecular substances on the surface of the thalli, and meanwhile, the betaine and tertiary butanol also have the effects of oxidation resistance, oxygen isolation and granulation enhancement and are not easy to break.

Optionally, the embedding agent comprises the following components in percentage by mass of 100 percent: trehalose 5 to 25% (e.g., 6%, 7%, 8%, 9%, 10%, 15%, 20%, 21%, 22%, 23% or 24%, etc.), betaine 0.1 to 1.0% (e.g., 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8% or 0.9%, etc.), t-butanol 0.5 to 5% (e.g., 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 4.5%, 4.8% or 4.9%), phospholipid 0.5 to 2% (e.g., 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.5%, 1.6%, 1.7%) 1.8% or 1.9%, etc.), 0.1 to 2% dibutyl phthalate (0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 1%, 1.5%, 1.6%, 1.7%, 1.8% or 1.9%, etc.), 5 to 10% sodium polyacrylate (e.g., may be 6%, 7%, 8% or 9%, etc.), 0.5 to 5% beta-cyclodextrin (e.g., may be 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4.5%, 4.6%, 4.7%, 4.8%, 4.9%, etc.), 1 to 5% hydroxypropyl starch (e.g., may be 1.5%, 2%, 2.5%, 3%, 3.5%, 4% or 4.5%, etc.), the balance being water.

Optionally, the mass ratio of the thallus to the embedding agent is 1 (1-5), for example, 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5, 1:4 or 1:4.5, etc.

Alternatively, the process may be carried out in a single-stage, the lactobacillus comprises one or more of streptococcus salivarius thermophilus, lactobacillus delbrueckii subsp bulgaricus, lactobacillus acidophilus, lactobacillus salivarius, lactobacillus helveticus, lactobacillus casei, lactobacillus paracasei, lactobacillus rhamnosus, lactobacillus plantarum, lactobacillus brevis, lactobacillus grignard, lactobacillus crispatus, lactobacillus fermentum, lactobacillus reuteri, lactobacillus johnsonii, lactobacillus jensenii, lactobacillus sake, lactobacillus curvatus, lactobacillus lactis subsp lactis, bifidobacterium longum, bifidobacterium breve, bifidobacterium animalis subsp adolescentis, bifidobacterium longum subsp infantis, bifidobacterium bifidum, bifidobacterium animalis subsp lactis, lactococcus lactis, pediococcus acidilactici or pediococcus pentosaceus.

Alternatively, the streptococcus salivarius comprises streptococcus salivarius subsp thermophilus ST81 (s.alivarius subsp. Thermophilus ST 81) and the lactobacillus delbrueckii comprises lactobacillus delbrueckii subsp. Bulgaricum LB 42.

Optionally, the granulating treatment comprises granulating the mixed liquid by adopting a liquid nitrogen granulator, and specifically comprises dripping the mixed liquid into liquid nitrogen through a dripping disc.

According to the invention, a liquid nitrogen granulation process is adopted, so that the material can be instantaneously reduced to the liquid nitrogen temperature (-196 ℃), the low-temperature freezing time of the thalli is shortened, meanwhile, uniform granular liquid drops can be prepared through a liquid drop tray, sublimation drying is directly carried out, the drying period is shortened, the production cost is reduced, and the solubility of the leavening agent is increased.

Optionally, the granulating treatment condition is that the aperture of a liquid dropping disc is 1-5 mm, and the feeding flow rate is 0.5-5 kg/h.

Optionally, the drying process comprises a vacuum freeze drying process.

Optionally, the conditions of the vacuum freeze-drying treatment are:

1) setting the vacuum degree to be 0.10+/-0.05 mbar, maintaining for 10-60 min, 2) setting the temperature in a drying cavity to be-35-10 ℃, maintaining the vacuum degree to be 0.13+/-0.05 mbar for 120-1200 min, 3) setting the temperature in the drying cavity to be 20-40 ℃ and maintaining the vacuum degree to be 0.13+/-0.05 mbar for 120-600 min.

Optionally, the method for collecting the thalli comprises the step of taking fermentation liquor and centrifuging for 10-20min at the temperature of 2-8 ℃ at 5000-8000 rpm.

The invention can also utilize vacuum aluminum thin bags for packaging and storage, further isolate the contact of thalli and oxygen and further improve the storage activity of the ferment.

As a preferred technical scheme, the preparation method of the yogurt starter comprises the following steps:

(1) Fermenting and culturing lactobacillus, detecting at least one of dielectric constant, viable count, fermentation activity or dry weight of thallus of fermentation liquid in the fermentation process, judging the fermentation end point, centrifuging the fermentation liquid at 5000-8000 rpm and 2-8 ℃ for 10-20 min, and collecting thallus;

(2) Mixing thalli and an embedding agent for 10-30 min at the temperature of 2-8 ℃ and the rpm of 1000-5000 to obtain a mixed solution according to the mass ratio of 1 (1-5), wherein the embedding agent contains trehalose, betaine, tertiary butanol, phospholipid, dibutyl phthalate, sodium polyacrylate, beta-cyclodextrin and hydroxypropyl starch;

(3) And granulating and drying the mixed solution by adopting a liquid nitrogen granulator to obtain the yogurt starter.

Compared with the prior art, the invention has at least the following beneficial effects:

The invention designs a preparation process of the yogurt starter, which can timely and accurately monitor the growth state of thalli in the reaction culture process through real-time online detection, rapidly and timely process the thalli to ensure the activity of the thalli to be in an optimal state, designs a specific thalli embedding agent, efficiently protects the thalli, designs a specific liquid nitrogen granulation process, shortens the low-temperature freezing time of the thalli, can prepare uniform granular liquid drops through a liquid drop tray, directly carries out sublimation drying, shortens the drying period, simultaneously can reduce the production cost, increases the solubility of the starter, and has good solubility, activity, fermentation activity and stability.

Drawings

FIG. 1 is a graph showing the linear relationship between dielectric constant (pF/cm) and fermentation activity.

FIG. 2 is a graph showing the results of the parameter change in the fermentation process.

FIG. 3 is a diagram showing the morphology of the starter.

FIG. 4 is a graph showing the results of storage stability of the starter.

Detailed Description

The technical scheme of the invention is further described below by the specific embodiments with reference to the accompanying drawings. The following examples are merely illustrative of the present invention and are not intended to represent or limit the scope of the invention as defined in the claims.

The specific techniques or conditions are not identified in the examples and are described in the literature in this field or are carried out in accordance with the product specifications. The reagents or equipment used were conventional products available for purchase by regular vendors, with no manufacturer noted.

In the specific embodiment of the invention, the trehalose is purchased from Texas Huiyang biotechnology Co., ltd, the betaine is purchased from Jinan Asia pharmaceutical industry Co., ltd, the tertiary butanol is purchased from Dongming Yuan Zhen Yuan industry Co., ltd, the phospholipid is purchased from Shandong Rui Kong chemical Co., ltd, the sodium polyacrylate is purchased from Henan Ming Chuan food technology Co., beta-cyclodextrin is purchased from Anhui Datang biological engineering Co., ltd, and the hydroxypropyl starch is purchased from Qingdao Hai Weisen biological technology Co., ltd.

The formulation of the common reagents used in the present invention is as follows, and will not be described in detail in the following examples.

MRS culture medium comprises dissolving peptone 10.00 g, beef extract 10.00 g, yeast extract 5.00 g, diammonium hydrogen citrate 2.00 g, glucose 20.00 g, tween-80 1mL, dipotassium hydrogen phosphate 2.00 g, manganese sulfate 0.58 g and magnesium sulfate 0.28 g in 1L distilled water, and adjusting pH to 6.4.

MRS solid culture medium 2% agar powder is added on the basis of MRS liquid culture medium.

Streaking and inoculating streptococcus salivarius thermophilus subspecies ST81 into an MRS solid culture medium, carrying out anaerobic culture for 48-72 hours at 37 ℃ to obtain single colonies, inoculating the prepared single colonies into an MRS liquid culture medium, and carrying out culture for 9-12 hours at 37 ℃ to activate;

Inoculating the bacterial liquid after 2 generations of activation into a 1L MRS liquid culture medium with an inoculum size of 2% (v/v), shaking and uniformly mixing, culturing in an anaerobic incubator at 37 ℃, linearly fitting the fermentation activity and an online living cell value, detecting an electric extremum through the online living cell in the fermentation process, characterizing the fermentation activity level of the bacterial body in real time, and when the bacterial body is cultured to the late stage of the logarithmic phase for 3-12 hours, timely processing the bacterial body in a state of more vigorous growth (the fermentation activity is best), higher activity (the bacterial liquid has the highest viable cell number) and highest bacterial body yield (the bacterial body dry weight is highest), centrifuging for 10-20 min at 5000~8000 rpm,2~8 ℃, removing the supernatant for later use, and preparing the streptococcus salivarius thermophilus ST81 bacterial body for later use.

The online living cell detection electrode value specifically refers to detection of fermentation broth by using online real-time monitoring equipment (equipment name: INCYTE ARC Expert; manufacturer: hamiltonian; model: 120), and the unit of detection dielectric constant is pF/cm.

Mixing the obtained streptococcus salivarius thermophilus ST81 thallus with embedding substances in a ratio of 1:1-5, wherein the mixing condition is that the mixture is mixed for 10-30 min at 1000-5000 rpm under the condition of 2-8 ℃, the embedding substances comprise, by mass, 5-25% of trehalose, 0.1-1.0% of betaine, 0.5-5% of tertiary butanol, 0.5-2% of phospholipid, 0.1-2% of dibutyl phthalate, 5-10% of sodium polyacrylate, 0.5-5% of beta-cyclodextrin, 1-5% of hydroxypropyl starch and the balance of water, the sterilization condition is that the mixture is sterilized for 10-60 min at 80-121 ℃, and the mixture is cooled to normal temperature for use, so that the embedding emulsion is finally obtained.

Granulating the obtained embedding mixture by a liquid nitrogen granulator, uniformly dripping the emulsion into liquid nitrogen through a dripping disc, discharging the produced particles by adopting a screw, and rapidly placing the particles into a vacuum drying cavity for vacuum drying.

The granulation condition is that the aperture of the liquid dropping disc is 1-5 mm, and the feeding flow rate is 2-5 kg/h.

The vacuum drying conditions are (1) the vacuum degree is set to be 0.10+/-0.05 mbar and maintained for 10-60 min, (2) the temperature in a drying cavity is set to be minus 35 to minus 10 ℃, the vacuum degree is set to be 0.13+/-0.05 mbar and maintained for 120-1200 min, and (3) the temperature in the drying cavity is set to be 25-40 ℃ and the vacuum degree is set to be 0.13+/-0.05 mbar and maintained for 120-600 min, so that the dried starter is prepared;

Different fermenting agents are prepared into fermenting agents with different characteristics by adjusting the proportion of the embedding formula and the liquid nitrogen granulation process according to the embedding formula and the liquid nitrogen granulation process of the strain. The other processes are the same.

In addition, the conventional preparation process is supplemented, namely, the streptococcus salivarius thermophilus ST81 bacterial cells obtained through fermentation are uniformly mixed and stirred with the prepared liquid freeze-drying protective agent (trehalose 15%, sucrose 4%, skimmed milk powder 6% and the balance of water) according to the proportion of 1:2 (m: m), vacuum freeze drying is directly carried out (the conditions are that the temperature is 1-50 ℃ to-45 ℃ for 3-4 h, the temperature is increased to-25 ℃ to-15 ℃ for 2) for 240-360 min, the temperature is maintained for 840-960 min, the temperature is increased to 0-5 ℃ for 3-6 h, the temperature is increased to 10-15 ℃ for 120-240 h, the temperature is maintained for 120-240 min for 5) for 60-90 min, the temperature is increased to 25 ℃ to 28 ℃ for 240-360 min, and the conventional process powder starter is obtained through crushing.

The number of live Streptococcus salivarius ST81 was determined by referring to the "food safety national Standard food microbiology test lactic acid bacteria test" (GB 4789.35-2016).

Fermentation activity of streptococcus salivarius thermophilus ST81 is that the streptococcus salivarius thermophilus ST81 is taken to ferment in sterilized fresh cow milk, pure milk and milk powder, the fermentation condition is that the fermentation is 6 h at 43 ℃, and the final pH value is recorded.

Storage stability of streptococcus salivarius thermophilus ST81, namely placing streptococcus salivarius ST81 particles prepared by adopting a cryogenic liquid nitrogen process at-20 ℃ and 4 ℃, sampling and detecting the viable count level of the streptococcus salivarius ST81 particles at 1 month, 6 months, 12 months, 18 months and 24 months in the process.

Streptococcus salivarius thermophilus ST81 drying survival (%)

The dry weight (DCW, g/L) of the cells of Streptococcus salivarius thermophilus ST81 was collected by centrifuging Streptococcus salivarius thermophilus ST81 (6500 rpm,15 min), followed by washing twice with physiological saline, and the obtained cells were dried in an oven to constant weight (drying condition: 105 ℃ C.) and weighed.

Example 1

This example shows the preparation of Streptococcus salivarius thermophilus ST81 cells.

And (3) streaking and inoculating streptococcus salivarius thermophilus subspecies ST81 into an MRS solid culture medium, carrying out anaerobic culture for 48-72 hours at 37 ℃ to obtain single colonies, inoculating the prepared single colonies into an MRS liquid culture medium, and carrying out culture for 3-12 hours at 37 ℃ to activate.

Inoculating the bacterial liquid after 2 generations of activation into a 1L MRS liquid culture medium with an inoculum size of 2% (v/v), shaking and uniformly mixing, culturing in an anaerobic incubator at 37 ℃, linearly fitting the value of an online living cell electrode with fermentation activity (pH) (figure 1) in the process, enabling the fitting R side to reach 0.99 with good linear relation, synchronously detecting the online living cell electrode directly in real time (figure 2) in the fermentation process until the period of 7-8 hours after the logarithmic phase (as shown in figure 2), processing the bacterial liquid in time in a state of relatively vigorous growth (the fermentation activity is the best), relatively high activity (the bacterial liquid has the highest viable count) and the highest bacterial yield (the bacterial dry weight is the highest), centrifuging 15 min under the condition of 8000 rpm and 4 ℃ for standby after removing the fermentation liquid, and preparing streptococcus salivarius thermophilus ST81 bacterial for standby.

Example 2

Different schemes are designed, the streptococcus salivarius thermophilus ST81 bacterial cells obtained in the embodiment 1 are mixed with embedding substances in a proportion of 1 (0.5-5), the mixing conditions are that under the condition of 4 ℃,3000 rpm is mixed for 20 min, the embedding substances comprise 5-25% of trehalose, 0.1-1.0% of betaine, 0.5-5% of tertiary butanol, 0.5-2% of phospholipid, 0.1-2% of dibutyl phthalate, 5-10% of sodium polyacrylate, 0.5-5% of beta-cyclodextrin and 1-5% of hydroxypropyl starch, residual water (sterilization conditions are that the sterilization is performed for 10-60 min at 80-121 ℃) is performed, the embedding emulsion is finally obtained, the particle starter is obtained through liquid nitrogen granulation and vacuum drying, and the influence of different schemes on the viable count of the particle starter and the fermentation activity of the streptococcus salivarius thermophilus ST81 are analyzed.

The first scheme is that the ratio of streptococcus salivarius thermophilus ST81 thallus to embedding substance is 1:0.5, the embedding substance comprises 8% of trehalose, 0.5% of betaine, 1.0% of tertiary butanol, 1.0% of phospholipid, 0.5% of dibutyl phthalate, 6% of sodium polyacrylate, 1.0% of beta-cyclodextrin, 2% of hydroxypropyl starch and the balance of water. Other conditions were consistent.

The second scheme is that the ratio of streptococcus salivarius thermophilus ST81 thallus to embedding substance is 1:1.5, the embedding substance comprises 8% of trehalose, 0.5% of betaine, 1.0% of tertiary butanol, 1.0% of phospholipid, 0.5% of dibutyl phthalate, 6% of sodium polyacrylate, 1.0% of beta-cyclodextrin, 2% of hydroxypropyl starch and the balance of water. Other conditions were consistent.

The third scheme is that the ratio of streptococcus salivarius thermophilus ST81 thallus to embedding substance is 1:5, the embedding substance comprises 8% of trehalose, 0.5% of betaine, 1.0% of tertiary butanol, 1.0% of phospholipid, 0.5% of dibutyl phthalate, 6% of sodium polyacrylate, 1.0% of beta-cyclodextrin, 2% of hydroxypropyl starch and the balance of water. Other conditions were consistent.

The fourth scheme is that the ratio of the streptococcus salivarius thermophilus ST81 thallus to the embedding substance is 1:1.5, the embedding substance comprises 15% of trehalose, 0.5% of betaine, 2.5% of tertiary butanol, 0.5% of phospholipid, 0.1% of dibutyl phthalate, 5% of sodium polyacrylate, 2.0% of beta-cyclodextrin, 3% of hydroxypropyl starch and the balance of water. Other conditions were consistent.

The fifth scheme is that the ratio of streptococcus salivarius thermophilus ST81 thallus to embedding substance is 1:1.5, the embedding substance comprises 15% of trehalose, 0.5% of betaine, 2.5% of tertiary butanol, 1.0% of phospholipid, 0.5% of dibutyl phthalate, 6% of sodium polyacrylate, 0.5% of beta-cyclodextrin, 1% of hydroxypropyl starch and the balance of water. Other conditions were consistent.

The ratio of the streptococcus salivarius thermophilus ST81 thallus to the embedding substance is 1:1.5, the embedding substance comprises 15% of trehalose, 0.5% of betaine, 2.5% of tertiary butanol, 1.0% of phospholipid, 0.5% of dibutyl phthalate, 6% of sodium polyacrylate, 4.5% of beta-cyclodextrin, 4% of hydroxypropyl starch and the balance of water. Other conditions were consistent.

The seventh scheme is that the ratio of streptococcus salivarius thermophilus ST81 thallus to embedding matter is 1:1.5, the embedding matter comprises trehalose 15%, betaine 0.5%, tertiary butanol 2.5%, phospholipid 1.0%, dibutyl phthalate 0.5%, sodium polyacrylate 6%, beta-cyclodextrin 1.0%, hydroxypropyl starch 3%, and the balance water. Other conditions were consistent.

The results of different schemes are shown in table 1, along with the addition of embedding substances, the number of living bacteria is improved to a certain extent, after the number of living bacteria reaches a certain value, the number of bacteria in each gram can be diluted due to too much embedding substances, so that the number of living bacteria is low, and the number of living bacteria and the freeze-drying survival rate can be influenced to a certain extent by the addition of different embedding substances in an embedding formula.

TABLE 1

Example 3

The embedding emulsion obtained in example 2 (prepared according to the scheme seven in example 2) is granulated by a liquid nitrogen granulator, the emulsion is uniformly dripped into liquid nitrogen through a dripping disc, and granules produced by discharging are quickly placed into a vacuum drying cavity for vacuum drying.

The basic scheme is as follows:

the granulation condition is that the aperture of the dropping disk is 2 mm, and the feeding flow rate is 2 kg/h;

the vacuum drying conditions are (1) the vacuum degree is set to be 0.10+/-0.05 mbar and maintained at 60 min, (2) the temperature in a drying cavity is set to be-30 ℃, the vacuum degree is set to be 0.13+/-0.05 mbar and maintained at 1200 min, and (3) the temperature in the drying cavity is set to be 30 ℃, the vacuum degree is set to be 0.13+/-0.05 mbar and maintained at 180 min, so that the dried streptococcus salivarius subspecies thermophilus ST81 particles are prepared.

The first scheme is that the granulation condition is that the aperture of a dropping disc is 1 mm, the feeding flow rate is 2 kg/h, and other conditions are consistent with the basic scheme.

The second scheme is that the granulation condition is that the aperture of a dropping disk is 2 mm, the feeding flow rate is 3 kg/h, and other conditions are consistent with the basic scheme.

The third scheme is that the granulation condition is that the aperture of a dropping disk is 5 mm, the feeding flow rate is 5 kg/h, and other conditions are consistent with the basic scheme.

The fourth scheme is that the vacuum drying condition is (1) the vacuum degree is set to be 0.10+/-0.05 mbar and maintained at 60 min, (2) the temperature in the drying cavity is set to be-30 ℃, the vacuum degree is set to be 0.13+/-0.05 mbar and maintained at 1200 min, and (3) the temperature in the drying cavity is set to be 25 ℃, the vacuum degree is set to be 0.13+/-0.05 mbar and maintained at 120 min, and other conditions are consistent with the basic scheme.

The fifth scheme is that the vacuum drying condition is (1) the vacuum degree is set to be 0.10+/-0.05 mbar and maintained at 60 min, (2) the temperature in the drying cavity is set to be-30 ℃, the vacuum degree is set to be 0.13+/-0.05 mbar and maintained at 1200 min, and (3) the temperature in the drying cavity is set to be 35 ℃, the vacuum degree is set to be 0.13+/-0.05 mbar and maintained at 60 min, and other conditions are consistent with the basic scheme.

The sixth scheme is that the vacuum drying condition is (1) the vacuum degree is set to be 0.10+/-0.05 mbar and maintained to be 30min, (2) the temperature in the drying cavity is set to be-20 ℃, the vacuum degree is set to be 0.13+/-0.05 mbar and maintained to be 950 min, and (3) the temperature in the drying cavity is set to be 30 ℃, the vacuum degree is set to be 0.13+/-0.05 mbar and maintained to be 300 min, and other conditions are consistent with the basic scheme.

The seventh scheme is that the vacuum drying condition is (1) the vacuum degree is set to be 0.10+/-0.05 mbar and maintained to be 30 min, (2) the temperature in the drying cavity is set to be-20 ℃, the vacuum degree is set to be 0.13+/-0.05 mbar and maintained to be 950 min, and (3) the temperature in the drying cavity is set to be 40 ℃, the vacuum degree is set to be 0.13+/-0.05 mbar and maintained to be 100 min, and other conditions are consistent with the basic scheme.

The scheme eight is that the vacuum drying condition is (1) the vacuum degree is set to be 0.10+/-0.05 mbar and maintained to be 60 min, (2) the temperature in the drying cavity is set to be-10 ℃, the vacuum degree is set to be 0.13+/-0.05 mbar and maintained to be 920 min, and (3) the temperature in the drying cavity is set to be 40 ℃, the vacuum degree is set to be 0.13+/-0.05 mbar and maintained to be 600 min, and other conditions are consistent with the basic scheme.

The vacuum drying condition is (1) that the vacuum degree is set to be 0.10+/-0.05 mbar and 60 min is maintained, (2) that the temperature in the drying cavity is set to be-10 ℃, the vacuum degree is set to be 0.13+/-0.05 mbar and 950 min is maintained, and (3) that the temperature in the drying cavity is set to be 25 ℃, the vacuum degree is set to be 0.13+/-0.05 mbar and 500 min is maintained, and other conditions are consistent with the basic scheme.

The streptococcus salivarius thermophilus ST81 particles obtained by the liquid nitrogen preparation process (scheme seven) are shown in figure 3, the particle preparation results of different schemes are shown in table 2, the size of the granulating particle size and the moisture content of the particles can be influenced by different liquid drop tray pore sizes and feeding flow rates, the larger the liquid drop tray pore size is, the larger the granulating particle size is, and meanwhile, the drying period and the moisture content can be influenced by different drying curves.

TABLE 2

Example 4

The streptococcus salivarius thermophilus subspecies ST81 prepared under the same conditions is taken and the starter is prepared by adopting a conventional process.

The advantage of using the liquid nitrogen preparation process of scheme seven of example 3 over conventional processes is as follows:

(1) Process variation

As shown in Table 3, the preparation process of the invention requires liquid nitrogen ultralow temperature treatment for thalli in the early stage, but the conventional process does not need liquid nitrogen ultralow temperature treatment, but the prefreezing time of the conventional process is 2.5-4.5 hours longer than that of the liquid nitrogen preparation process, and the drying period of the preparation process of the invention is shortened by 18-21 hours averagely than that of the conventional process in terms of drying time, so that the production cost can be greatly reduced for industrial scale production. Meanwhile, the starter prepared by the preparation process is granular, is more soluble in water than the starter prepared by the conventional process, and has no caking and floating phenomenon.

TABLE 3 Table 3

(2) Freeze-drying survival rate

As shown in Table 4, the viable count of the ferment prepared by the process is improved by 113% and the survival rate is improved by 95% compared with the conventional process, and the process uses an instantaneous low-temperature environment to quickly reduce the temperature of the ferment from a normal temperature state to a low-temperature (-196 ℃) state, and shortens the low-temperature pre-freezing time, so that the damage of the formation of ice crystals to cell membranes of the ferment is reduced, the survival of the ferment after freeze-drying is facilitated, and the viable count and the survival rate of the ferment are further improved.

TABLE 4 Table 4

(3) Fermentation activity level

The invention and the conventional process are adopted to ferment in sterilized fresh cow milk, pure milk and milk powder, the fermentation condition is that the fermentation is 6 h under the temperature of 43 ℃, the pH value change result is recorded as shown in the table 5, the pH change of different milk sources fermented by the ferment prepared by the process is slightly faster than that of the conventional ferment, and the pH change of the fermented milk powder of two dosage forms is faster than that of the fermented fresh milk and the pure milk.

TABLE 5

The curding time of the starter for milk powder fermentation, the viable count of the yoghurt after 4 ℃ refrigeration of 12h, the yoghurt texture, aroma and taste were tested, and the specific results are shown in table 6.

TABLE 6

As can be seen from Table 6, the curding time of the starter of the process of the present invention was significantly faster than that of the conventional process under the same inoculum size and fermentation conditions, and there was no significant difference between the viable count of yogurt and the organoleptic flavor of the yogurt after refrigeration at 4℃of 12 h. The starter of the process has less damage in the freeze-drying process, increased cell number for maintaining complete activity, and shortened curd time, and the starter of the process has no change in the fermentation metabolism path of strain, so that the sensory flavor of yoghurt is the same as that of conventional process.

(4) Post acid stability level

The acidity of the yogurt samples after the end of the 43 ℃ fermented milk powder with different ferment was examined after the yogurt samples were placed at 4 ℃ for cold storage for 7 days, 14 days and 21 days, and the acidity was detected by an acid-base titration method, and the specific experimental results are shown in table 7.

TABLE 7

As is clear from the results in Table 7, there was no significant difference in post-acid change between the two fermenters, and there was no gradient decrease in post-acid change with time of the fermenters, and it was found that the fermenters of the process of the present invention had no effect on post-acid of the fermented yogurt. The post-acid is influenced by factors such as strain, fermentation environment and the like, the starter of the process is only innovated in the process of preparing the starter, and the characteristics and metabolic pathways of the strain are not influenced, so that the post-acid and acidity change is not influenced.

(5) Storage stability and fermentation viability level

The results of the sampling and detecting the cell survival rate of the 1 ST month, 6 th month, 12 th month, 18 th month and 24 th month in the process of placing the different leavening agents packaged by the aluminum thin bags for two years at-18 ℃,4 ℃ and 25 ℃ respectively are shown as the results shown in figure 4, the survival rate of the different process leavening agents is reduced to a certain extent after the leavening agents are stored for two years at-18 ℃,4 ℃ and 25 ℃, the storage survival rate of the leavening agents in the process is not obvious compared with the conventional process, the storage survival rate of the leavening agents in the process in the 4 ℃ and 25 ℃ is obviously better than that in the conventional process (p < 0.05), wherein the survival rate of streptococcus salivarius thermophilus subspecies ST81 in the 4 ℃ and 25 ℃ is respectively improved by 13.8% and 36.4% compared with that in the conventional process, and the storage stability of the leavening agents in the process is better.

Fermenting the stored starter under different conditions in sterilized milk powder under the conditions of initial pH 6.5+ -0.1,43 deg.C fermenting 6 h and other conditions being consistent, recording pH value at 6 h of fermentation, and Table 8 shows the results.

TABLE 8

After the starter is stored for different time, the starter is stored for 2 years at the temperature of-18 ℃ and the fermentation activity is not obvious compared with that of the starter in the conventional process, and the starter is stored at the temperature of 4 ℃ and 25 ℃ and is obviously superior to that of the starter in the conventional process (p < 0.05). The process provided by the invention has the advantages that the damage to the obtained ferment is small in the freeze-drying process, the number of cells which keep complete activity is increased, and the fermentation activity stability of the thalli is also improved to a certain extent.

In summary, the method can accurately monitor the growth state of the thalli in the reaction culture process in time by carrying out real-time online detection through the online living cell detection electrode, rapidly and timely process the thalli to ensure the activity of the thalli to be in an optimal state, design a specific thalli embedding agent, efficiently protect the thalli, design a specific liquid nitrogen granulation process, instantly reduce the temperature (-196 ℃) of materials to liquid nitrogen, shorten the low-temperature freezing time of the thalli, simultaneously prepare uniform granular liquid drops through a liquid drop tray, directly carry out sublimation drying, reduce the production cost and increase the dissolution performance of the ferment, and further isolate the contact between the thalli and oxygen by adopting a vacuum aluminum thin bag for packaging and storage, thereby further improving the storage activity of the ferment. The starter prepared by the process has good solubility, activity, fermentation activity and stability.

The applicant declares that the above is only a specific embodiment of the present invention, but the scope of the present invention is not limited thereto, and it should be apparent to those skilled in the art that any changes or substitutions that are easily conceivable within the technical scope of the present invention disclosed by the present invention fall within the scope of the present invention and the disclosure.

Claims (10)

1. A method for preparing a yogurt starter, the method comprising:

Fermenting and culturing lactobacillus, detecting at least one of dielectric constant, viable count, fermentation activity or dry weight of thallus of fermentation liquid in fermentation process, judging fermentation end point, and collecting thallus;

mixing thalli with an embedding agent to obtain a mixed solution, and granulating and drying the mixed solution to obtain the yogurt starter;

The embedding agent contains trehalose, betaine, tertiary butanol, phospholipid, dibutyl phthalate, sodium polyacrylate, beta-cyclodextrin and hydroxypropyl starch.

2. The preparation method of the yogurt starter according to claim 1, wherein the embedding agent comprises, by mass, 100% of trehalose 5-25%, betaine 0.1-1.0%, t-butanol 0.5-5%, phospholipid 0.5-2%, dibutyl phthalate 0.1-2%, sodium polyacrylate 5-10%, beta-cyclodextrin 0.5-5%, hydroxypropyl starch 1-5%, and the balance water.

3. The method for producing a yogurt starter according to claim 1, wherein the mass ratio of the bacterial cells to the embedding medium is 1 (1-5).

4. The method for producing a lactic acid bacterium starter according to claim 1, wherein the lactic acid bacterium comprises one or more of streptococcus salivarius thermophilus subspecies bulgaricus, lactobacillus acidophilus, lactobacillus salivarius, lactobacillus helveticus, lactobacillus casei, lactobacillus paracasei, lactobacillus rhamnosus, lactobacillus plantarum, lactobacillus brevis, lactobacillus grignard, lactobacillus crispatus, lactobacillus fermentum, lactobacillus myxomatosus, lactobacillus reuteri, lactobacillus johnsonii, lactobacillus jensenii, lactobacillus sake, lactobacillus curvatus, lactobacillus lactis subspecies lactis, bifidobacterium longum subspecies longum, bifidobacterium breve, bifidobacterium animalis subspecies subsp. Adolescentis, bifidobacterium longum subsp. Infantis, bifidobacterium animalis subsp. Animalis, lactobacillus subsp. Lactis, pediococcus acidilactis, or pediococcus pentosaceus.

5. The method for producing a yogurt starter according to claim 1, characterized in that the granulating treatment comprises granulating the mixed liquid with a liquid nitrogen granulator, specifically comprising dropping the mixed liquid into liquid nitrogen through a dropping tray.

6. The method for preparing a yogurt starter according to claim 5, wherein the granulating treatment is carried out under the conditions that the diameter of a drip tray is 1-5 mm and the feeding flow rate is 0.5-5 kg/h.

7. The method for producing a yogurt starter for use according to claim 1, characterized in that the drying treatment comprises a vacuum freeze-drying treatment.

8. The method for producing a yogurt starter for use according to claim 7, wherein the conditions of the vacuum freeze-drying treatment are:

1) setting the vacuum degree to be 0.10+/-0.05 mbar, maintaining for 10-60 min, 2) setting the temperature in a drying cavity to be-35-10 ℃, maintaining the vacuum degree to be 0.13+/-0.05 mbar for 120-1200 min, 3) setting the temperature in the drying cavity to be 20-40 ℃ and maintaining the vacuum degree to be 0.13+/-0.05 mbar for 120-600 min.

9. The method for preparing a yogurt starter according to claim 1, wherein the method for collecting the bacterial cells comprises centrifuging the fermentation liquid at 5000-8000 rpm and 2-8deg.C for 10-20 min.

10. The method for producing a yogurt starter for use according to any one of claims 1 to 9, characterized in that the method comprises the steps of:

(1) Fermenting and culturing lactobacillus, detecting at least one of dielectric constant, viable count, fermentation activity or dry weight of thallus of fermentation liquid in the fermentation process, judging the fermentation end point, centrifuging the fermentation liquid at 5000-8000 rpm and 2-8 ℃ for 10-20 min, and collecting thallus;

(2) Mixing thalli and an embedding agent for 10-30 min at the temperature of 2-8 ℃ and the rpm of 1000-5000 to obtain a mixed solution according to the mass ratio of 1 (1-5), wherein the embedding agent contains trehalose, betaine, tertiary butanol, phospholipid, dibutyl phthalate, sodium polyacrylate, beta-cyclodextrin and hydroxypropyl starch;

(3) And granulating and drying the mixed solution by adopting a liquid nitrogen granulator to obtain the yogurt starter.