A Lactobacillus sakei room-temperature-set carrot yogurt and its preparation method

Abstract

The present invention belongs to the field of microbial technology, and particularly relates to a Lactobacillus sakei room-temperature coagulated carrot yogurt and a preparation method thereof. The present invention provides a preparation method of room-temperature coagulated carrot yogurt, and the preparation method comprises the following steps: mixing carrot juice and milk evenly, adding fermentable sugar to obtain a mixed solution; homogenizing and performing high-temperature sterilization on the mixed solution, and cooling to room temperature to obtain a sterilized solution; adding a starter to the sterilized solution for fermentation to obtain room-temperature coagulated carrot yogurt. The preparation method provided by the present invention has low cost and significantly shortens the preparation time; the prepared yogurt has a good curd state, a unique flavor and a delicate taste.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbiology, and particularly relates to a Lactobacillus sakei room-temperature solidified carrot yogurt and a preparation method thereof. Background Art

[0002] Probiotic yogurt has gradually been accepted by the general consumers due to its stable quality, good safety, unique flavor and health care value (Zheng Min, Du Jingjing, Lu Yi, Bai Yuyan. Research progress on the application of functional yogurt [J]. Modern Food, 2022, 28(13): 48-50.). A large number of studies have shown that probiotics have multiple health care effects on the human body, such as regulating the intestinal flora, maintaining the balance within the flora, promoting food digestion, improving constipation, reducing cholesterol levels, alleviating lactose intolerance, inhibiting the proliferation of harmful bacteria and the production of harmful substances in the intestine, and inhibiting the aging of the body. Thus, it can be seen that the physiological functions of probiotics are very beneficial to the life activities of the body.

[0003] Research has shown that the fermentation temperature has a greater impact on the quality of yogurt (Lyn, C, Radke-Mitchell, et al. Influence of Temperature on Associative Growth of Streptococcus thermophilus and Lactobacillus bulgaricus [J]. Journal of Dairy Science, 1986, 54(1): 17-22.; Meybodi N M, Mortazavian A M, Arab M, et al. Probiotic viability in yoghurt: A review of influential factors [J]. International Dairy Journal, 2020, 109(1): 104-793.). Currently, the main fermentation strains of yogurt are Lactobacillus thermophilus, Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus. By making a yogurt starter with these conventional fermentation strains in a certain proportion, yogurt can be made at home by controlling the temperature at 37-42°C with a yogurt maker. Therefore, the current home fermentation of yogurt depends on the temperature control of fermentation equipment (such as a home yogurt maker), but the yogurt maker needs to be purchased separately, which will incur additional costs.

[0004] Chinese Patent Application No. 202111577737.6 discloses a preparation method of yogurt, comprising the following steps: mixing milk and white sugar, homogenizing and sterilizing, then inoculating Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus in an inoculation amount ratio of 6:1-1:1, fermenting at 44-45°C, and when the fermentation reaches the end point, the end point pH≤4.5 and the acidity range is 70-80°T to obtain the yogurt.

[0005] Chinese Patent Application No. 202111659262.5 discloses a method for preparing yogurt, which includes: fermenting raw milk and / or reconstituted milk with a sweetener, Xinjiang tomato paste, an acidity regulator, a starter culture, and GABA-producing synergistic bacteria to obtain fermented milk; mixing a stabilizer with water, and then mixing the obtained liquid with the fermented milk; the fermentation temperature is 28-30 °C and the time is 24-36 hours.

[0006] The quality of yogurt fermented at room temperature is higher than that of yogurt fermented at high temperature. Moreover, yogurt fermented at room temperature has a more delicate and smooth taste after coagulation, has a unique aroma of fermented milk, and has less whey separation (Meybodi N M, Mortazavian A M, Arab M, et al. Probiotic viability in yoghurt: A review of influential factors[J]. International Dairy Journal, 2020, 109(1): 104-793.; Choi Y J, Jin H Y, Yang H S, et al. Quality and storage characteristics of yogurt containing Lacobacillus sakei ALI033 and cinnamon ethanol extract[J]. Journal of Animal Science & Technology, 2016, 58(1): 16.). During the ripening and refrigeration stages, the acidity of yogurt fermented at room temperature is more stable than that of yogurt fermented at high temperature, and the changes in acidity and pH during post-ripening are small. In addition, the fat content of yogurt fermented at room temperature is slightly higher than that of yogurt fermented at high temperature (Ali, Mostafaie, Gholamreza, et al. Effect of fermentation temperature and different Streptococcus thermophilus to Lactobacillus bulgaricus ratios on Kermanshahi roghan and yoghurt fatty acid profiles.[J]. The Journal of dairy research, 2018, 23(3): 72-75.).

[0007] Therefore, there is an urgent need in the current field for a method to ferment yogurt at room temperature to save fermentation costs and improve the quality of yogurt. Summary of the Invention

[0008] In the present invention, considering the health benefits of the preparation raw materials and the needs of consumers, carrots are selected as the preparation raw materials. At the same time, the inventor unexpectedly isolated a strain of Lactobacillus sakei (also known as Lactiplantibacillus sakei) CGMCC No. 27270 from pickled Chinese cabbage. This Lactobacillus sakei has good fermentation characteristics and can grow at room temperature in milk, making it suitable for room-temperature yogurt fermentation (A R P L, A M J M, A C A P, et al. Physicochemical and microbial changes in yogurts produced under different pressure and temperature conditions. ScienceDirect[J]. LWT, 2019, 99(3): 423 - 430.; Marceau A, Zagorec M, Marie-Christine Champomier-Vergès. Positive effects of growth at suboptimal temperature and high salt concentration on long-term survival of Lactobacillus sakei. [J]. Research in Microbiology, 2003, 154(1): 37 - 42.).

[0009] The technical solution for the present invention to achieve the above technical objectives is as follows:

[0010] Preservation Information:

[0011] Biological Material: MH-LS-01

[0012] Taxonomic Name: Lactiplantibacillus sakei

[0013] Preservation Date: May 6, 2023

[0014] Preservation Location: General Microbiological Center of China National Culture Collection Center

[0015] Preservation Number: CGMCC No. 27270

[0016] One of the objectives of the present invention is to provide a method for preparing room-temperature set carrot yogurt, and the method includes the following steps:

[0017] (1) Mix the carrot juice and milk evenly, and then add fermentable sugar to obtain a mixed solution;

[0018] (2) Homogenize and sterilize the mixed solution at a high temperature, and cool it to room temperature to obtain a sterilized solution;

[0019] (3) Add a fermenting agent to the sterilized solution for fermentation to obtain room-temperature solidified carrot yogurt.

[0020] In some embodiments, step (1) further includes:

[0021] Crush and filter the carrots to obtain carrot juice.

[0022] In some embodiments, the fermentable sugar includes, but is not limited to, glucose, fructose, sucrose, lactose, maltose or maltotriose.

[0023] In some preferred embodiments, the fermentable sugar is sucrose or maltose, preferably sucrose.

[0024] In some embodiments, the mass-volume ratio (g / mL) of the fermentable sugar is 5%-7%, where g is the mass of the fermentable sugar and mL is the total volume of the carrot juice and milk.

[0025] In some preferred embodiments, the mass-volume ratio of the fermentable sugar is 6%.

[0026] In some embodiments, the conditions for the high-temperature sterilization in step (2) are 85°C for 5 minutes.

[0027] In some embodiments, the conditions for the homogenization in step (2) include:

[0028] 1) The pressure is 220-250 bar;

[0029] 2) The temperature is 60-65°C.

[0030] In some embodiments, the fermentation in step (3) is carried out at a constant temperature.

[0031] The fermentation needs to be carried out under aseptic conditions, and those skilled in the art can select a specific aseptic environment according to common general knowledge and conventional technical means.

[0032] In some embodiments, the fermentation in step (3) is carried out at a constant temperature of 22°C-28°C.

[0033] In some embodiments, the fermentation in step (3) can be carried out at a constant temperature of 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, or 28°C.

[0034] In some preferred embodiments, the fermentation in step (3) is a constant temperature fermentation at 25°C.

[0035] In some embodiments, the fermentation time in step (3) is 14-18 hours.

[0036] In some embodiments, the fermentation time in step (3) is 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, or 18 hours.

[0037] In some preferred embodiments, the fermentation time in step (3) is 16 hours.

[0038] In some embodiments, step (3) further comprises:

[0039] After the fermentation stops, the fermentation liquid is obtained and refrigerated for post-ripening.

[0040] In some preferred embodiments, the refrigerated post-ripening condition is standing at 4° C. for 10-20 hours.

[0041] In some embodiments, the acidity of the fermentation broth is 67.3-68.3°F.

[0042] In some embodiments, the viable cell count of the fermentation broth is at least 1.1×10 9 CFU / mL.

[0043] In some embodiments, the fermentation culture comprises mesophilic lactic acid bacteria.

[0044] In some embodiments, the mesophilic lactic acid bacteria include, but are not limited to, Lactobacillus plantarum, Lactobacillus paracasei, Lactobacillus sakei, or Lactobacillus curvatum.

[0045] In some embodiments, the starter culture comprises at least 7.5×10 7 CFU / mL mesophilic lactic acid bacteria.

[0046] In some preferred embodiments, the mesophilic lactic acid bacteria may be Lactobacillus sakei.

[0047] In some preferred embodiments, the mesophilic lactic acid bacteria may be CGMCC No. 27270 Lactobacillus sakei.

[0048] In some embodiments, the fermentation culture comprises 7.68×10 7 -1.31×10 8 CFU / mL of CGMCCNo.27270 sake Lactobacillus.

[0049] In some embodiments, the fermentation culture comprises 1.00×10 8-1.08×10 8 CFU / mL of *Lactobacillus sakei* CGMCC No. 27270.

[0050] In some preferred embodiments, the starter culture contains 1.00×10 8 , 1.01×10 8 , 1.02×10 8 , 1.03×10 8 , 1.04×10 8 , 1.05×10 8 , 1.06×10 8 , 1.07×10 8 , 1.08×10 8 CFU / mL of *Lactobacillus sakei* CGMCC No. 27270.

[0051] In some preferred embodiments, the starter culture contains 1.04×10 8 CFU / mL of *Lactobacillus sakei* CGMCC No. 27270.

[0052] The second object of the present invention is to provide a room-temperature set carrot yogurt prepared by any of the foregoing methods.

[0053] In some preferred embodiments, the room-temperature set carrot yogurt is a *Lactobacillus sakei* room-temperature set carrot yogurt.

[0054] In some preferred embodiments, the room-temperature set carrot yogurt is a *Lactobacillus sakei* CGMCC No. 27270 room-temperature set carrot yogurt.

[0055] In some embodiments, the acidity of the room-temperature set carrot yogurt is 67.3 - 68.3 °T, preferably 67.8 °T.

[0056] In some embodiments, the viable count of the room-temperature set carrot yogurt is at least 1.1×10 9 CFU / mL, preferably 1.1×10 9 CFU / mL.

[0057] In some embodiments, the water holding rate of the room-temperature set carrot yogurt is at least 40%.

[0058] In some embodiments, the pH value of the room-temperature set carrot yogurt is greater than 4.3, preferably 4.66 - 4.68.

[0059] In some embodiments, the room-temperature coagulable carrot yogurt further comprises food-acceptable excipients, including but not limited to, gelatin, pectin, agar, carrageenan, gellan gum, modified starch (e.g., hydroxypropyl distarch phosphate), whey protein, milk protein, concentrated milk protein, cream, edible flavor, high fructose syrup, granulated sugar, sodium carboxymethyl cellulose, citric acid, sodium citrate, diglycerol fatty acid ester, etc.

[0060] A third object of the present invention is to provide a probiotic product, which comprises any one of the foregoing room-temperature coagulable carrot yogurts and a container for loading the room-temperature coagulable carrot yogurt.

[0061] In some embodiments, the container can be formed of a variety of materials, such as glass or plastic. The container contains a probiotic ferment, which includes the room-temperature coagulable carrot yogurt itself and / or other probiotic ferments (e.g., thermophilic lactobacillus yogurt, bulgarian lactobacillus yogurt).

[0062] The probiotic product can maintain the biological activity of active ingredients (e.g., mesophilic lactobacillus, preferably lactobacillus sakei), and does not contain additional ingredients that are unacceptably toxic to the subject to whom the probiotic product will be administered or consumed.

[0063] The probiotic product is sterile.

[0064] Compared with the prior art, the preparation method of the lactobacillus sakei room-temperature coagulable carrot yogurt provided by the present invention has at least the following technical effects:

[0065] (1) Realize room-temperature fermentation, get rid of the dependence on fermentation equipment, and significantly save the preparation cost;

[0066] (2) Significantly shorten the fermentation time. Most of the prior art requires fermentation for more than 20 h, and the present invention only needs 16 h at the fastest to complete fermentation;

[0067] (3) The prepared yogurt has excellent taste and flavor, and the sensory score is as high as 90.5, which is increased by 15.6% compared with the control group (bacterial powder group);

[0068] (4) The prepared yogurt has appropriate acidity, higher water holding rate, and higher viable bacteria count;

[0069] (5) Higher apparent viscosity, which is more conducive to maintaining the solidified state. BRIEF DESCRIPTION OF THE DRAWINGS

[0070] Figure 1 Shows the effect of the addition amount of bacterial strains on the titratable acidity of yogurt.

[0071] Figure 2 Shows the effect of the addition amount of bacterial strains on the sensory score of yogurt.

[0072] Figure 3 It shows the influence of fermentation time on the viable count of yogurt.

[0073] Figure 4 It shows the influence of fermentation time on the titratable acidity of yogurt.

[0074] Figure 5 It shows the influence of fermentation time on the sensory score of yogurt.

[0075] Figure 6 It shows the influence of fermentation temperature on the viable count of yogurt.

[0076] Figure 7 It shows the influence of fermentation temperature on the titratable acidity of yogurt.

[0077] Figure 8 It shows the influence of fermentation temperature on the sensory score of yogurt.

[0078] Figure 9 It shows the influence of sucrose addition amount on the viable count of yogurt.

[0079] Figure 10 It shows the influence of sucrose addition amount on the titratable acidity of yogurt.

[0080] Figure 11 It shows the influence of sucrose addition amount on the sensory score of yogurt.

[0081] Figure 12 and Figure 13 It shows the influence of the interaction between different factors (fermentation time and strain inoculation amount) on the sensory evaluation.

[0082] Figure 14 and Figure 15 It shows the influence of the interaction between different factors (fermentation temperature and strain inoculation amount) on the sensory evaluation.

[0083] Figure 16 and Figure 17 It shows the influence of the interaction between different factors (fermentation temperature and fermentation time) on the sensory evaluation.

[0084] Figure 18 It shows the influence of different starters on the apparent viscosity of yogurt.

[0085] Figure 19 It shows the influence of different starters on the sensory evaluation of yogurt. Detailed implementation mode

[0086] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly used in the field to which this invention belongs. For the purpose of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural and vice versa.

[0087] Unless the context clearly indicates otherwise, the expressions "a" and "an" as used herein include plural referents. For example, reference to "a cell" includes a plurality of such cells and equivalents known to those skilled in the art and the like.

[0088] The term "about" as used herein means a range of ±20% of the value that follows. In some embodiments, the term "about" means a range of ±10% of the value that follows. In some embodiments, the term "about" means a range of ±5% of the value that follows.

[0089] The numerical ranges used herein should be understood to have enumerated all the numbers within that range. For example, the range of 1 to 20 should be understood to include any number, combination of numbers, or sub-range from the following group: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

[0090] The term "comprises" or "comprising" as used herein means "including but not limited to". This term is intended to be open-ended, to specify the presence of any stated feature, element, integer, step, or component, but not to exclude the presence or addition of one or more other features, elements, integers, steps, components, or groups thereof. Thus, the term "comprising" includes the more restrictive terms "consisting of" and "consisting essentially of". In one embodiment, the term "comprising" used throughout the application, particularly in the claims, may be replaced by the term "consisting of".

[0091] The terms "optionally", "either", "any", or "any one" as used herein mean that the subsequent described event or circumstance may but need not occur, and this description includes the instances where the event or circumstance occurs or does not occur.

[0092] The term "and / or" as used herein should be understood to mean any one of the alternatives or any combination of two or more of the alternatives.

[0093] The term "normal temperature" as used herein refers to room temperature or indoor temperature, i.e., the indoor environmental temperature in a natural state without the aid of any instruments and / or equipment. In some instances, normal temperature is 20°C - 28°C. In some preferred instances, normal temperature is 24°C - 26°C. In some more preferred instances, normal temperature is 26°C.

[0094] As used herein, the terms "Latilactobacillus sakei", "Latilactobacillus sakei CGMCC No.27270", or "Latilactobacillus sakei" refer to lactic acid bacteria that are Gram-positive, negative in the catalase test, with a colony morphology of white, raised, opaque, smooth surface, and regular colony edges, and a strain morphology of short rods, arranged singly or in pairs. In some examples, the Latilactobacillus sakei is deposited in the China General Microbiological Culture Collection Center (CGMCC), and its deposit number is CGMCC No.27270. In some examples, the Latilactobacillus sakei may also be referred to as Lactobacillus sakei var. sakei. Those skilled in the art should know that both of these names refer to Latilactobacillus sakei, preferably Latilactobacillus sakei CGMCC No.27270.

[0095] As used herein, the term "mesophilic lactic acid bacteria" refers to a class of lactic acid bacteria with an optimal growth temperature of 20-40 °C, and its main characteristic is the production of diacetyl, acetaldehyde, and CO2 by citrate fermentation.

[0096] To make the objectives, technical solutions, and advantages of the present invention clearer and more understandable, the following further details the present invention in combination with examples. For those not specified in the examples, conventional conditions or conditions recommended by the manufacturer are followed. All reagents or instruments not indicating the manufacturer can be obtained as conventional products through commercial purchase. To better illustrate the present invention, numerous specific details are given in the following specific embodiments. The specific embodiments described herein are only used to explain the present invention and do not constitute any limitation to the present invention. In addition, in the following description, the description of well-known structures and technologies is omitted to avoid unnecessarily confusing the concepts of the present invention. Such structures and technologies are also described in many publications, such as "Molecular Cloning: A Laboratory Manual (Fourth Edition)" (Cold Spring Harbor Laboratory Press), Ausubel, F.M. et al., Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley-Interscience.

[0097] Table 1 Main reagents and materials involved in the present invention

[0098]

[0099]

[0100] The experimental methods of the present invention:

[0101] · pH value determination: Measured with a pH meter, three parallel samples are measured each time, and the results are averaged.

[0102] · Acidity determination: Refer to GB 5009.239-2016 "Determination of Food Acidity", and the titration acidity method is used. Three parallel samples are measured each time, and the results are averaged.

[0103] · Viable count determination: Refer to GB 4789.35-2016 "National Food Safety Standard Food Microbiology Examination Lactic Acid Bacteria Examination" for the determination of viable counts.

[0104] · Water holding capacity determination: Weigh 15-30 g of ripened yogurt into a centrifuge tube, set the centrifugation conditions to 4000 r / min and centrifuge at 4 °C for 20 min. After centrifugation, remove the supernatant and weigh the mass of the remaining precipitate. Three parallel samples are measured for each sample, and the arithmetic mean of the results is taken (Wang Xinlei, Wang Meng, Yan Chunxiao, Geng Weitao, Wang Yanping. Effects of Lactobacillus kefiranofaciens ZW3 on the flavor and texture of yogurt [J]. Food Science, 2022, 4(10): 119-123.). The calculation formula for the water holding capacity of yogurt is as follows:

[0105]

[0106] · Rheological property determination: The rheological properties of yogurt are determined by the small amplitude frequency sweep method. First, stir the sample clockwise 10 times, and then counterclockwise 10 times. A stainless steel plate probe with a diameter of 50 mm is selected, and the gap between the plate and the bottom surface is 1 mm. The test temperature is controlled at 25 ± 0.5 °C. Take an appropriate amount of yogurt and place it on the substrate of the rotational rheometer for measurement. The specific test conditions are as follows: a plate with a diameter of 50 mm, the temperature is set at 25 °C, the gap distance is adjusted to 1 mm, and the shear rate of the yogurt rises from 0.1 s -1 to 100 s -1 within 5 min (Lei Yonggang. Study on the rheological properties and microstructure of soy yogurt [D]. South China University of Technology, 2013.).

[0107] · Sensory evaluation: A 15-member panel of food professionals conducts sensory evaluation on yogurt from five aspects: color, texture, odor, taste, and degree of preference. The scores are taken three times and averaged. The evaluation criteria are shown in Table 2:

[0108] Table 2 Reference table for sensory evaluation of yogurt

[0109]

[0110]

[0111] Experimental data processing of the present invention:

[0112] Excel 2016 and SPSS26.0 software were used for data statistics, Origin 2019b software was used for graphing, and Design-Expert8.0.6.1 software was used to design the response surface experiment and analyze the data.

[0113] The preparation method of the room temperature solidified yogurt of the present invention:

[0114] Wash fresh carrots, crush them with a wall-breaking machine, and filter through gauze to obtain carrot juice;

[0115] 20% (v / v, ratio of the volume of carrot juice to the volume of milk) carrot juice was mixed with 80% (v / v, ratio of the volume of milk to the volume of carrot juice) milk and 6% (v / w, ratio of the mass of sucrose to the total volume of carrot juice and milk) sucrose for preparation;

[0116] Homogenize (220-250 bar pressure, 60-65°C), sterilize at 85°C for 5 minutes, then cool to room temperature;

[0117] Inoculate 1.04×10 8 CFU / mL sake bacillus MH-LS-01 culture;

[0118] After mixing, ferment at a constant temperature of 25°C for 16 hours.

[0119] After fermentation, the product was refrigerated and ripened (standing at 4°C for 10-20 hours) to obtain room temperature solidified carrot yogurt.

[0120] Example 1 Activation and preparation of bacterial strains

[0121] Lactobacillus sakei MH-LS-01 was activated and inoculated into MRS broth medium. After inoculation, it was placed in an incubator and cultured for 12 hours. It was diluted in a clean bench to make the number of lactic acid bacteria 10 8 CFU / mL, lactic acid bacteria were inoculated into MRS liquid culture medium at a ratio of 3%, and cultured at 20°C for 3 days to obtain sake-spreading Lactobacillus MH-LS-01 culture, which can be subsequently prepared into bacterial agent, bacterial powder or freeze-dried powder for use as needed.

[0122] Example 2 Preparation of Normal Temperature Coagulation Type Carrot Yogurt

[0123] Wash fresh carrots, crush them with a wall-breaking machine, and filter through gauze to obtain carrot juice;

[0124] Mix 200mL of carrot juice with 800mL of milk and 60g of sucrose;

[0125] · Homogenize (at a pressure of 220 - 250 bar and a temperature of 60 - 65 °C), sterilize at 85 °C for 5 min, and then cool to room temperature;

[0126] · Inoculate 1.04×10 8 CFU / mL of Lactobacillus sakei MH-LS-01 culture under aseptic conditions;

[0127] · After mixing, ferment at a constant temperature of 25 °C for 16 h.

[0128] · After fermentation, carry out cold storage and after-ripening (stand at 4 °C for 10 - 20 h) to obtain room-temperature coagulated carrot yogurt.

[0129] Example 3 Effect of Strain Addition Amount on the Quality of Room-Temperature Coagulated Carrot Yogurt

[0130] The preparation method of the yogurt in this example is the same as that in Example 2. To control the single-factor variable, only the addition amount of the strain is changed (the addition amounts of the strain in each group of experiments are 10 7.72 (5.24×10 7 )CFU / mL, 10 7.89 (7.68×10 7 )CFU / mL, 10 8.02 (1.04×10 8 )CFU / mL, 10 8.12 (1.31×10 8 )CFU / mL, 10 8.20 (1.57×10 8 )CFU / mL).

[0131] The results show that:

[0132] As the addition amount of the strain increases, the acidity of the yogurt gradually increases, and the pH value of the yogurt continuously decreases ( Figure 1 );

[0133] The sensory score of the yogurt first increases and then decreases with the increase of the addition amount of the strain. When the addition amount of the strain is 1.04×10 8 CFU / mL, the sensory score of the yogurt is the highest, and the yogurt has a fine texture, good color and luster, and a sweet and sour taste; when the addition ratio of the strain is lower than 1.04×10 8 CFU / mL, the fermentation process is slow, the fermentation is incomplete, the acid production rate is slow, the acidity of the yogurt is insufficient, and it presents a semi-solid state with inconsistent structure, affecting the quality of the yogurt; when the addition ratio of the strain is higher than 1.04×10 8 CFU / mL, the acid production rate and the pH value decrease rate accelerate ( Figure 2 ).

[0134] Therefore, considering the acidity value and sensory score of the yogurt, a fermentation bacterium inoculation amount of 7.68×10 7 -1.31×10 8 CFU / mL was selected for the response surface experiment.

[0135] Example 4: Effect of Fermentation Time on the Quality of Room Temperature Coagulated Carrot Yogurt

[0136] The preparation method of the yogurt in this example was the same as that in Example 2. To control the single-factor variable, only the fermentation time was changed (the fermentation times of each group of experiments were 12h, 14h, 16h, 18h, and 20h).

[0137] The results showed that:

[0138] As the fermentation time increased, the viable count of Lactobacillus sakei and the acidity of the yogurt both increased, and the pH value of the yogurt continuously decreased ( Figure 3 and Figure 4 );

[0139] The sensory score of the yogurt showed a trend of first increasing and then decreasing as the fermentation time increased. When the fermentation time was 16h, the sensory score of the yogurt was the highest, and the yogurt had a fine tissue state, good color and luster, and a sweet and sour taste; when the fermentation time was less than 16h, the fermentation time was too short, resulting in uneven color of the yogurt, weak thickening feeling, and uneven structure, affecting the quality of the yogurt; when the fermentation time was higher than 16h, the fermentation time was too long, resulting in too sour taste of the yogurt, causing the sensory score to decrease ( Figure 5 ).

[0140] Therefore, considering the acidity value and sensory score of the yogurt, a fermentation time of 14h - 18h was selected for the response surface experiment.

[0141] Example 5: Effect of Fermentation Temperature on the Quality of Room Temperature Coagulated Carrot Yogurt

[0142] The preparation method of the yogurt in this example was the same as that in Example 2. To control the single-factor variable, only the fermentation temperature was changed (the fermentation temperatures of each group of experiments were 19°C, 22°C, 25°C, 28°C, and 31°C).

[0143] The results showed that:

[0144] As the fermentation temperature increased, the viable count of Lactobacillus sakei and the acidity of the yogurt both increased, and the pH value of the yogurt continuously decreased ( Figure 6 and Figure 7 );

[0145] The sensory score of the yogurt first increases and then decreases with the increase of the fermentation temperature. When the fermentation temperature is 25 °C, the sensory score of the yogurt is the highest, reaching the maximum value. At this time, the yogurt curd is uniform, with a strong fragrance and a delicate and smooth taste. When the fermentation temperature is lower than 25 °C, the fermentation process is slow, the acid production rate is slow, the yogurt has no yogurt aroma, and there are bubbles on the surface, affecting the quality of the yogurt. When the fermentation temperature is higher than 25 °C, the acid production rate and the pH value decrease too fast, the acidity of the yogurt is too high, and the color is uneven( Figure 8 ).

[0146] Example 6 Effect of Sucrose Addition on the Quality of Room Temperature Coagulated Carrot Yogurt

[0147] The preparation method of the yogurt in this example is the same as that in Example 2. To control the single-factor variable, only the sucrose addition amount is changed (the sucrose addition amounts in each experimental group are respectively 2%, 4%, 6%, 8%, and 10%).

[0148] With the increase of the sucrose addition amount, the viable count of Lactobacillus sakei in the yogurt and the yogurt acidity both show a trend of first increasing and then decreasing, and the pH value of the yogurt shows a trend of first decreasing and then increasing( Figure 9 and Figure 10 );

[0149] The sensory score of the yogurt first increases and then decreases with the increase of the sucrose addition amount. When the sucrose addition amount is 6%, the sensory score of the yogurt is the highest, and the yogurt has a fine texture, good color, and a sweet and sour taste. When the sucrose addition ratio is lower than 6%, the taste is too sour and the acceptance is not high. When the sucrose addition ratio is higher than 6%, the sweet and sour ratio of the yogurt is out of balance and the public preference decreases( Figure 11 ).

[0150] Example 7 Optimization of Fermentation Process by Response Surface Experiment

[0151] On the basis of the single-factor test results, taking the inoculum amount, fermentation temperature, and fermentation time as response variables and the sensory score as the response value, a three-factor and three-level Box-Behnken test was carried out using Design Expert 8.0.6 to optimize the fermentation process of the best starter culture for yogurt. The levels of each factor in the test are shown in Table 3.

[0152] Table 3 Experimental Factor Level Coding Table

[0153]

[0154] 7.1 Response Surface Experiment Design

[0155] A three-factor and three-level Box-Behnken test was carried out using Design Expert8.0.6. The test design and results are shown in Table 4.

[0156] Table 4 Response Surface Experiment Design and Results

[0157]

[0158] 7.2 Establishment and Significance Analysis of the Model

[0159] Based on the results in Table 4, Design-Expert software was used for fitting the binary regression equation and variance analysis. The regression equation obtained is as follows: Y = 33.54 - 1.35A - 1.27B + 0.44C - 0.35AB - 0.48AC + 0.038BC - 2.31A 2 - 1.99B 2 - 0.71C 2 , and the results of variance analysis are shown in Table 5.

[0160] Table 5 Variance Analysis Table of the Regression Equation

[0161]

[0162]

[0163] Note: **: indicates extremely significant (p < 0.01); *: indicates significant (p < 0.05)

[0164] As can be seen from Table 5, the quadratic polynomial analysis model in Table 2 shows that the regression of this model is extremely significant (p < 0.01), indicating that the model fits the actual experiment well; the lack-of-fit error term is not significant (p > 0.05), indicating that the predicted values and actual values of the model are in good agreement; and the R 2 of this model is 0.9991, indicating that 99.91% of the variation in the response value of this model is due to the selected variables, further indicating that the model has a good fitting degree and the optimal technological conditions of yogurt can be predicted through this model. In the regression equation, A, C, BC, A 2 , B 2 [[ID=3�]]C 2 have an extremely significant impact on the sensory evaluation of yogurt (p < 0.01), and B, AB have a significant impact on the polysaccharide yield (p < 0.05). The larger the F value, the greater the impact of the corresponding factor on the experimental index. As can be seen from Table 5, the order of the impact on the sensory evaluation of yogurt is: fermentation time > fermentation temperature > strain addition amount.

[0165] 7.3 Response Surface Analysis of the Interaction between Factors

[0166] Using Design-Expert 8.0.6.1 software, response surface analysis diagrams ( Figures 12 - 14 ) were generated. Analyze the impact of the interaction between factors on the sensory evaluation.

[0167] The results show that the interaction of various factors in the experiment can be illustrated by the response surface and contour lines. The results of the effects of fermentation time, fermentation temperature, or inoculum addition amount on the sensory evaluation of yogurt all increase first and then decrease, and the response values show a parabolic trend, indicating that there is a maximum value in the regression equation. The contour lines are close to ellipses, indicating a significant interaction between time and inoculum addition amount, which is consistent with the analysis results of the interaction term values in Table 5( Figure 12 ). The contour lines are close to circles, indicating that the interaction between fermentation time and fermentation temperature has no significant effect( Figure 13 ). The interaction between fermentation temperature and inoculum addition amount is significant, which is consistent with the analysis results in Table 5( Figure 14 ). It can be seen from this that the response surface method can be used to optimize the fermentation yogurt process.

[0168] Using Design-Expert to predict the optimal process conditions for yogurt fermentation are a fermentation temperature of 25.8 °C, a fermentation time of 15.39 h, and an inoculum addition amount of 9.9×10 7 CFU / mL. The predicted sensory score is 91.5, and the viable count is 1.1×10 9 CFU / mL. For the convenience of experimental operation, after rounding the optimized conditions, the obtained fermentation temperature is 25 °C, the fermentation time is 16 h, and the inoculum addition amount is 1.04×10 8 CFU / mL.

[0169] From the response surface optimization analysis, the optimal process conditions for room temperature fermented yogurt are: a fermentation time of 16 h, an inoculum addition amount of 1.04×10 8 CFU / mL, and a temperature of 25 °C. At this time, the sensory score is 90.5 points.

[0170] Example 8 Effect of Starter on Rheological Properties of Room Temperature Set Carrot Yogurt

[0171] In this example, different starters were selected for fermentation to explore the effect of starters on the rheological properties of room temperature set carrot yogurt.

[0172] The preparation method of room temperature set carrot yogurt is the same as that in Example 2, only changing the type of starter.

[0173] The starters used in this example include: bacterial powder (i.e., Jiajiale Yogurt Fermentation Powder IV, which contains freeze-dried powder of Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus rhamnosus, Streptococcus thermophilus, Bifidobacterium infantis, Bifidobacterium longum, Lactobacillus casei, and Lactobacillus acidophilus); L. sakei CGMCC No. 27270 of the present invention.

[0174] The results show that the apparent viscosities of the two groups of yogurts are in the range of 0.1 - 100 s -1Within the shear range, it shows a decreasing trend with the increase of shear rate, indicating that yogurt has the property of shear thinning. Shear thinning means that the apparent viscosity of solidified yogurt gradually decreases with the increase of shear rate. During the process of increasing shear rate, the apparent viscosity of Lactobacillus sakei group yogurt is greater than that of the bacterial powder group at the initial shear, and then they tend to be the same ( Figure 15 ).

[0175] Example 9 Effect of Starter Cultures on the Sensory Properties of Room Temperature Set Carrot Yogurt

[0176] In this example, different starter cultures were used for fermentation to explore the effect of starter cultures on the sensory properties of room temperature set carrot yogurt.

[0177] The preparation method of room temperature set carrot yogurt was the same as that in Example 2, only changing the type of starter culture.

[0178] The starter cultures used in this example included: bacterial powder (the same as in Example 8); Lactobacillus sakei CGMCC No. 27270 of the present invention.

[0179] The results showed that the sensory scores of yogurt in the bacterial powder group and the Lactobacillus sakei group were 90.5 and 78.3 respectively ( Figure 16 ). Compared with the bacterial powder group, the yogurt in the Lactobacillus sakei CGMCC No. 27270 group had a more delicate and smooth taste, a unique aroma of fermented milk, and less whey separation, and significantly improved the sensory score of yogurt (p < 0.05). Compared with the bacterial powder group, the sensory score of yogurt in the Lactobacillus sakei CGMCC No. 27270 group increased by 15.6%. The yogurt in the Lactobacillus sakei CGMCC No. 27270 group had no negative effects on smell and taste.

[0180] Comparative Example Effect of Starter Cultures on the Quality of Room Temperature Set Carrot Yogurt

[0181] In this comparative example, different starter cultures were used for fermentation to explore the effect of starter cultures on the quality of room temperature set carrot yogurt.

[0182] The preparation method of room temperature set carrot yogurt was the same as that in Example 2, only changing the type of starter culture.

[0183] The starter cultures used in this example included: bacterial powder (the same as in Example 8); Lactobacillus sakei CGMCC No. 27270 of the present invention; Lactobacillus sakei ATCC 15521 (purchased from ATCC), and the results are shown in Table 6.

[0184] Table 6 Effect of Different Starter Cultures on the Quality of Yogurt

[0185]

[0186]

[0187] The pH of the yogurt with the bacterial powder group was 4.04, the acidity was 110.1 °T, the water holding capacity was 33.12%, and the viable count was 8.2×10 8 CFU / mL; the pH of the yogurt with Lactobacillus sakei group CGMCC No.27270 was 4.47, the acidity was 67.8 °T, the water holding rate was 41.7%, and the viable count was 1.1×10 9 CFU / mL; the pH of Lactobacillus sakei ATCC 15521 was 4.10, the acidity was 85.3 °T, and the water holding rate was 35%. Compared with the bacterial powder group and the conventional Lactobacillus sakei (ATCC 15521), it had a more suitable acidity, a higher water holding rate, and better quality.

[0188] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than limiting the protection scope of the present invention. Any simple modification or equivalent replacement of the technical solution of the present invention by those of ordinary skill in the art shall not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A preparation method of a room-temperature solidifying carrot yogurt, characterized in that, The preparation method comprises the following steps: (1) Mix carrot juice and milk evenly, and then add fermentable sugar to obtain a mixed solution; (2) Homogenize and sterilize the mixed solution at a high temperature, and cool it to room temperature to obtain a sterilized solution; (3) Add a starter to the sterilized solution for fermentation to obtain a room-temperature coagulated carrot yogurt. In step (1), the fermentable sugar is sucrose with a mass-volume ratio (g / mL) of 5%-7%, where g is the mass of the fermentable sugar and mL is the total volume of milk and carrots. In step (3), the fermentation is carried out at a constant temperature of 25 °C, and the fermentation time is 14 - 18 h; the fermenting agent is Lactobacillus sakei CGMCC No. 27270 with a concentration of 7.68×10 7 -1.31×10 8 CFU / mL.

2. The preparation method according to claim 1, characterized in that, The fermentation time in step (3) is 16 h.

3. The preparation method according to claim 1, characterized in that, The starter culture described in step (3) is Lactobacillus sakei CGMCC No. 27270 with a concentration of 1.00×10 8 -1.08×10 8 CFU / mL.

4. The preparation method according to claim 3, characterized in that, The starter culture described in step (3) is Lactobacillus sakei CGMCC No. 27270 at 1.04×10 8 CFU / mL.

5. The preparation method according to claim 1, characterized in that, The fermentable sugar is sucrose with a mass-volume ratio of 6%.

6. The preparation method according to claim 1, characterized in that, Step (3) further includes: After fermentation stops, the fermented liquid is obtained and subjected to cold storage and after-ripening, wherein the conditions for cold storage and after-ripening are standing at 4°C for 10-20 h.

7. A room-temperature coagulated carrot yogurt prepared by the preparation method according to any one of claims 1-6.

8. The room temperature solidifying type carrot yogurt according to claim 7, characterized in that, The room-temperature coagulated carrot yogurt further contains food-acceptable excipients, including at least one of gelatin, pectin, agar, carrageenan, gellan gum, modified starch, whey protein, milk protein, concentrated milk protein, light cream, edible essence, high fructose syrup, granulated sugar, sodium carboxymethyl cellulose, citric acid, sodium citrate, and diglycerol fatty acid ester.

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