A process for preserving bread with a lactic acid bacteria ferment instead of a preservative

Abstract

The present application relates to food microbial preservation and fresh-keeping technology, and discloses a bread preservation process method of lactic acid bacteria fermentation product which can replace preservatives. In the method, the cheese whey protein is fermented by Lactobacillus casei R-1, and the fermentation product obtained is freeze-dried and then added into toast bread at different concentrations to effectively inhibit the main spoilage microorganisms (including Escherichia coli, Staphylococcus aureus and mold) in the bread. At the same time, the method systematically evaluates the influence of the fermentation product addition on the texture and flavor of toast bread, significantly prolongs the bread preservation period, and does not significantly affect the taste and quality of the bread. Compared with the traditional chemical preservatives or refrigeration method, the present application uses natural microbial metabolites to achieve "clean label" preservation without adding chemical preservatives, which is of great significance to improve the safety and health properties of toast bread and provides a new idea for the natural preservation technology of baked foods.

Description

Technical Field

[0001] This invention relates to the field of food microbial preservation and freshness technology, and specifically discloses a process for preserving bread using lactic acid bacteria fermentation that can replace preservatives. Background Technology

[0002] Toast, a popular baked good, is a staple in daily life due to its portability and delicious taste. However, its high moisture content and rich nutrients make it highly susceptible to microbial contamination and spoilage, resulting in a short shelf life. Traditional preservation methods include adding chemical preservatives, vacuum packaging, and refrigeration. While chemical preservatives effectively extend shelf life, long-term consumption poses potential health risks, and their widespread use is increasingly restricted as consumers demand more natural and healthy foods. Vacuum packaging and refrigeration can delay spoilage to some extent, but they increase costs and energy consumption, and maintaining suitable low temperatures throughout sales and consumption is difficult, affecting preservation effectiveness.

[0003] In recent years, with the development of fermentation science, especially the in-depth application of microorganisms and probiotics in food preservation, using lactic acid bacteria fermentation technology to replace some preservatives and improve the preservation quality of bread has become a research hotspot. Lactic acid bacteria, as highly regarded probiotics, not only offer health benefits to humans but also play a crucial role in the global baking industry. Their fermentation techniques for sourdough (such as lactobacillus) have a long history in many European and American countries. For example, in Italy, more than 40 types of lactic acid bacteria, including *Lactobacillus plantarum* and *Lactobacillus curvilinearis*, have been isolated and identified in traditional wheat bread dough such as Cornetto, demonstrating the widespread application and important fermentation value of lactic acid bacteria in bread production.

[0004] While yeast remains the primary fermentation agent in domestic bread production, the fermentation and innovative applications of lactic acid bacteria are increasingly attracting attention. Lactic acid bacteria extracts, obtained through deep liquid fermentation methods (such as fermentation tank processes), possess the ability to reproduce the flavor and nutritional advantages of traditional sourdough fermentation. Adding lactic acid bacteria extracts to bread baking not only simplifies traditional mixed fermentation operations but also significantly increases bread volume, delays staling, improves texture and flavor, and extends shelf life. In some innovative companies both domestically and internationally, fermentation products rich in different lactic acid bacteria genera are gradually being promoted and implemented as preservative-free bread preservation solutions.

[0005] Although Western lactic acid bacteria sourdough technology is widely used, research and industrial application of using specific lactic acid bacteria ferments for preservation in the Chinese market, especially in the production of toast bread with high daily consumption, is still in the exploratory stage. Summary of the Invention

[0006] The present invention aims to provide a lactic acid bacteria fermentation technology for preserving bread that can replace preservatives, avoiding the health hazards, high costs, and increased energy consumption associated with other preservation methods.

[0007] To achieve the above objectives, the first aspect of this invention provides a method for preparing lactic acid bacteria ferment for bread preservation, comprising the following steps:

[0008] Lacticaseibacillus casei R-1 is provided, with accession number CGMCCNO.33871;

[0009] The Lactobacillus casei R-1 was inoculated into a culture medium containing whey protein for fermentation to obtain a fermentation broth;

[0010] The fermentation broth is post-processed to obtain the lactic acid bacteria fermentation product.

[0011] In some embodiments of the method described in the first aspect, the fermentation conditions include:

[0012] The fermentation temperature is 28-40℃, preferably 37℃;

[0013] The fermentation time is 16-120 hours, preferably 72 hours;

[0014] The inoculation amount is 3%-7% (v / v), preferably 5% (v / v).

[0015] In some embodiments of the method described in the first aspect, the post-processing includes:

[0016] Centrifuge the fermentation broth and collect the supernatant;

[0017] Optionally, the supernatant may be filtered to remove bacteria;

[0018] The supernatant was freeze-dried to obtain freeze-dried powder.

[0019] A second aspect of the present invention provides a lactic acid bacteria fermentation product prepared by any one of the methods described in the first aspect.

[0020] A third aspect of the present invention provides a bread containing the lactic acid bacteria fermentation product described in the second aspect.

[0021] In some specific embodiments of bread proposed in the third aspect, the amount of lactic acid bacteria ferment added is 0.3%-1.0% (w / w) based on 100% of the total flour mass, preferably 0.5%-0.8% (w / w).

[0022] In some specific embodiments of the bread proposed in the third aspect, the bread is toast.

[0023] The fourth aspect of this invention provides the use of the lactic acid bacteria fermentation product described in the second aspect in the preparation of baked goods.

[0024] In some of the usage embodiments proposed in the fourth aspect, the usage is for inhibiting microorganisms in baked goods and / or improving the flavor of baked goods and / or delaying the aging of baked goods.

[0025] In some of the usage embodiments proposed in the fourth aspect, the microorganisms include bacteria and / or molds, preferably one or more of Staphylococcus aureus, Escherichia coli and Aspergillus niger.

[0026] Advantages of the present invention

[0027] The prominent functions of lactic acid bacteria in bread preservation are mainly reflected in two aspects: First, their antibacterial and preservative effects. During fermentation, lactic acid bacteria produce various metabolites such as lactic acid and bacteriocins, which have significant broad-spectrum antibacterial effects, effectively inhibiting various pathogenic bacteria (such as Listeria, Staphylococcus aureus, and Bacillus subtilis) and common bread spoilage fungi (such as Penicillium, Aspergillus, and Anthracis). Second, they improve dough processing and enhance flavor. The fermentation products of lactic acid bacteria contain rich organic acids, enzymes, and aromatic components, which can improve the rheological properties of dough, enhance the delicate texture of bread, and delay aging. The protein hydrolysis system of lactic acid bacteria can break down proteins into more amino acids and small peptides, improving dough structure and promoting the release of soluble flavor substances, giving baked bread a richer and more natural flavor and a better texture. At the same time, the organic acids and other substances produced by lactic acid bacteria during fermentation can also improve the freshness and elasticity of bread, partially replacing chemical emulsifiers and further increasing the added value of the product.

[0028] The *Lactaseibacillus casei* R-1 used in this invention was deposited on March 19, 2025, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC NO. 33871. *Lactaseibacillus casei* R-1, as a lactic acid bacterium with good acid resistance and intestinal colonization ability, not only possesses certain health benefits from its fermentation products but also exhibits significant potential for food preservation and shelf-life extension due to the production of abundant antibacterial substances.

[0029] This invention discloses a lactic acid bacteria fermentation process for preserving bread, which can replace preservatives. Its core advantage lies in achieving clean bread production through the use of natural microbial metabolites. By employing *Lactobacillus casei* R-1 fermented whey protein, which has excellent antibacterial properties, this process not only significantly inhibits common spoilage microorganisms such as *Staphylococcus aureus*, *Escherichia coli*, and *Aspergillus niger*, extending the shelf life of bread to over 9 days with an extension rate exceeding 100%, but also exhibits superior antibacterial effects compared to traditional chemical preservatives like calcium propionate. Furthermore, this fermentation product effectively optimizes the texture of the dough, giving toast a finer and softer structure. By producing key flavor compounds such as limonene and ethyl 2-methylbutyrate, it significantly enhances the fruity and fermented ester aromas of bread without the addition of chemically synthesized flavorings, achieving significant advantages in preservation, texture quality, and natural flavor.

[0030] Therefore, developing a new preservation process for toast bread based on Lactobacillus casei R-1 fermentation material not only aligns with the food industry's consumer trend towards "natural, healthy, and low-additive" products, but also provides bread companies with a safe, effective, and economical new preservation strategy. This invention focuses on the preparation, antibacterial evaluation, and practical application of Lactobacillus casei R-1 fermentation material in bread. Through in vitro and bread-based validation, it has been demonstrated that the material possesses excellent antibacterial capabilities and extends shelf life, potentially providing a novel technological pathway for the widespread application of probiotic fermentation materials in the field of baked goods preservation. Attached Figure Description

[0031] Figure 1 Photographs showing the inhibition zone results of the fermentation supernatant of Lactobacillus casei R-1 against Staphylococcus aureus S1 (left) and Escherichia coli E (right);

[0032] Figure 2 Comparative photographs of Aspergillus niger growth on plates under the treatment of Lactobacillus casei R-1 fermentation supernatant (left) and pure water control (right);

[0033] Figure 3 shows the mold growth of toast bread with different concentrations of Lactobacillus casei R-1 fermentation product and blank control after being stored at room temperature.

[0034] Figure 4 Radar graph showing the effects of different concentrations of Lactobacillus casei R-1 fermentation product on the textural properties of toast bread (including hardness, elasticity, stickiness, chewiness, cohesion, and adhesion).

[0035] Figure 5 This is a two-dimensional scatter plot of principal component analysis (PCA) of the effects of different concentrations of Lactobacillus casei R-1 fermentation product on the flavor compounds of toast bread based on GC-IMS data.

[0036] Figure 6This is a heatmap showing the relative content of key volatile flavor compounds in toast bread of different concentrations of Lactobacillus casei R-1 fermentation products and the blank control group, based on GC-IMS data. Detailed Implementation

[0037] The following are specific embodiments of the present invention, which will further illustrate the technical solution of the present invention in detail, but the scope of protection of the present invention is not limited to these embodiments.

[0038] Lacticaseibacillus casei R-1 was deposited on March 19, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.33871.

[0039] Unless otherwise specified, all materials and reagents used in this invention are purchased from the open market.

[0040] Example 1: Preparation of Lactobacillus casei R-1 whey fermentation product

[0041] 1. Activation culture of *Lactobacillus casei* R: Under aseptic conditions, single colonies of *Lactobacillus casei* R-1 were picked using a sterile inoculation loop and inoculated into 50 mL of MRS medium. The cultures were then incubated for 18 hours in a constant temperature shaker at 37 ℃ and 180 rpm to obtain the first-generation seed culture. This process was repeated for two consecutive subcultures to obtain a stable third-generation seed culture.

[0042] 2. Preparation of fermentation medium: Accurately weigh 100 g of whey protein powder, dissolve it in 1000 mL of deionized water, stir well, and dispense into 250 mL Erlenmeyer flasks. Autoclave at 115 ℃ and 0.1 MPa for 20 minutes. After sterilization, cool to room temperature (approximately 25 ℃) for later use.

[0043] 3. Fermentation culture: In a sterile laminar flow hood, R-1 seed culture was inoculated into sterile whey protein medium at an inoculation rate of 5% (v / v). The mixture was cultured at 37°C and 180 rpm with shaking for 72 hours to obtain the R-1 fermentation broth.

[0044] 4. Obtaining fermentation supernatant: Centrifuge the above fermentation broth at 6000 rpm for 15 minutes and collect the supernatant. Then filter the centrifuged supernatant through a 0.22 μm microporous membrane to remove bacteria, thus obtaining the R-1 fermentation supernatant.

[0045] 5. Preparation of freeze-dried fermentation product powder: The supernatant of R-1 fermentation was freeze-dried, and the resulting freeze-dried block was ground to obtain freeze-dried R-1 whey fermentation product powder, which was sealed and dried at 4 ℃ for storage.

[0046] Example 2: Preparation of R-1 Whey Fermented Product Toast Bread

[0047] This embodiment provides a method for preparing toast bread with added R-1 whey starter.

[0048] 1. Formula: Based on 100% high-gluten flour, the remaining ingredients are: 60% water, 6% granulated sugar, 5% butter, 4% egg liquid, 1.5% yeast, 1% salt, 2% milk powder, and different weight percentages (0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%) of R-1 whey fermented protein freeze-dried powder. An unfermented whey protein group was set up as a blank control.

[0049] 2. Kneading: Add all dry ingredients (including R-1 leavening agent powder) and wet ingredients (except butter) to a dough mixer. Mix on low speed for 2 minutes, then switch to high speed and mix until the dough begins to develop gluten. Add the butter and continue mixing until the dough can be stretched into a thin, even film (fully developed stage).

[0050] 3. First fermentation: Place the dough in a fermentation box at 28℃ and 75% relative humidity for 60 minutes, until the dough volume expands to about twice its original size.

[0051] 4. Dividing and shaping: Divide the fermented dough into 150g portions, roll them into balls, let them rest for 20 minutes, then roll them out and place them in a loaf pan.

[0052] 5. Second fermentation: Place the loaf pan in a fermentation box at 35 ℃ and 85% relative humidity, and ferment until the dough reaches eight-tenths full of the loaf pan.

[0053] 6. Baking: Place in a preheated oven at 180°C and bake for 30 minutes.

[0054] 7. Cooling and Packaging: Immediately after baking, demold and cool at room temperature until the core temperature drops below 35°C, then seal in aseptic bags.

[0055] Experimental Example 1-2 Evaluation of the antibacterial properties of fermentation supernatant of Lactobacillus casei R-1

[0056] Experimental Example 1: Determination of Antibacterial Ability

[0057] To verify the inhibitory effect of Lactobacillus casei R-1 fermentation supernatant on common food spoilage bacteria, Staphylococcus aureus (Gram-positive) and Escherichia coli (Gram-negative) were selected as indicator bacteria. The specific experimental steps are as follows:

[0058] 1. Activation of bacterial strains: The preserved Staphylococcus aureus and Escherichia coli were inoculated into LB liquid medium and cultured with shaking at 37 ℃ and 180 rpm for 16 hours to restore their growth activity.

[0059] 2. Preparation of bacterial plates: Mix the activated bacterial solution with 0.5 mL of LB solid medium (50 mL) that has been autoclaved and cooled to about 50 °C, and quickly pour the mixture into sterile petri dishes to prepare uniform bacterial plates.

[0060] 3. Sample addition: Using a sterile punch, prepare two 10 mm diameter wells on each bacterial plate, and add 100 μL of R-1 fermentation supernatant sample to each well. As a control, add an equal volume of sterile pure water and 1% (w / w) calcium propionate to the wells of the bacterial plate.

[0061] 4. Incubation and Observation: Place the plate with the added sample in a 37℃ incubator and incubate for 24 hours. Observe whether an inhibition zone appears around the sample well, and accurately measure the diameter of the inhibition zone with calipers. Each group should have three replicates.

[0062] Table 1. Inhibitory effects of whey protein supernatant from different strains on food spoilage bacteria.

[0063]

[0064] Note: *Calcium propionate is a chemical preservative permitted under GB2760-2024 and has a recognized antibacterial effect, but its effect is weaker than that of R-1 fermentation supernatant.

[0065] The results are as follows Figure 1 As shown in Table 1, the R-1 fermentation supernatant exhibited significant antibacterial effects, demonstrating excellent inhibitory capabilities against both typical Gram-positive bacteria (Staphylococcus aureus) and Gram-negative bacteria (Escherichia coli). Both showed clear and regular inhibition zones, significantly superior to the chemical preservative calcium propionate. In contrast, the pure water control group showed no inhibition zones, fully demonstrating that the observed antibacterial effect originated entirely from the active metabolites in the R-1 fermentation supernatant, rather than from physical or operational factors. In conclusion, the fermentation supernatant of *Lactobacillus casei* R-1 possesses significant and broad-spectrum in vitro antibacterial capabilities, effectively inhibiting common food spoilage bacteria and pathogens, including *Staphylococcus aureus* and *Escherichia coli*. This lays a solid theoretical foundation for its subsequent application as a natural biological preservative in bread systems, replacing chemical preservatives.

[0066] Experiment Example 2: Determination of Antifungal Activity

[0067] To evaluate the inhibitory effect of R-1 fermentation supernatant on mold, the common putrefactive mold *Aspergillus niger* was selected as an indicator fungus. The specific experimental steps are as follows:

[0068] 1. Mold activation: Inoculate Aspergillus niger into PDA liquid culture medium and culture it at 28 ℃ under static conditions for 3 days to allow it to fully produce spores and grow.

[0069] 2. Preparation of sample plates: Prepare PDA solid culture medium, autoclave and cool to about 50 °C, add 1 mL of R-1 fermentation supernatant, an equal amount of sterile pure water and 1% (w / w) calcium propionate as a control, mix thoroughly and pour into plates to prepare sample plates and control plates.

[0070] 3. Inoculation and culture: Using a sterile punch, take 10 mm diameter Aspergillus niger cakes and inoculate them into the center of the above-mentioned plates. Place the plates in a 28 ℃ constant temperature incubator and incubate for 3 days.

[0071] 4. Results observation: Observe and compare the growth status of each group of molds, including morphological characteristics such as colony diameter, hyphal density, spore formation and colony color.

[0072] Table 2. Inhibitory effect of whey protein supernatant from different bacterial strains on Aspergillus niger.

[0073]

[0074] The results are as follows Figure 2 As shown in Table 2, compared with the control group, the growth of Aspergillus niger colonies in the fermentation supernatant treatment group was significantly inhibited, with smaller colony expansion, sparse hyphae, and significantly reduced spore formation; while in the control group, mold growth was vigorous, with large colony coverage, dense hyphae, dark black color, and abundant spore production. These results indicate that the R-1 fermentation supernatant has good antifungal activity.

[0075] Experimental Example 3-5: Study on the Preservative Effect of R-1 Whey Fermented Product on Toast Bread

[0076] Experimental Example 3: Detection of Microbial Indicators

[0077] 1. Sampling and Processing: The bread samples prepared in Example 2 were stored at room temperature (25±2℃). Samples were taken on days 0, 7, and 14 of storage. Under aseptic conditions, 1.0 g of bread crumb sample was weighed from each group, added to 9.0 mL of sterile physiological saline, homogenized for 2 minutes to prepare a 1:10 sample homogenate, and then serially diluted 10-fold to obtain a suitable colony concentration for counting.

[0078] 2. Microbial count:

[0079] (1) Total bacterial count: Select 2-3 suitable dilutions, take 100 μL of dilution solution, spread it on plate counting agar (PCA) plates, and incubate upside down in a 37 ℃ incubator for 48 hours.

[0080] (2) Staphylococcus aureus: Take 100 μL of dilution, spread it on Baird-Parker agar (BP) plates, and incubate upside down in a 37°C incubator for 24-48 hours.

[0081] (3) Coliform bacteria: Take 100 μL of the dilution and spread it on crystal violet neutral red bile salt agar (VRBA) plates. Incubate upside down in a 37°C incubator for 24 hours.

[0082] (4) Mold: Take 100 μL of the dilution solution, spread it on rose red sodium agar (RBA) plates, and incubate upside down in an incubator at 28℃ for 5-7 days.

[0083] 3. Colony counting and recording: After the culture is completed, the typical colonies that grow on various culture medium plates are counted, and the colony forming units (CFU / g) contained in each gram of bread sample are calculated according to the dilution factor.

[0084] Shelf life diagram:

[0085] Table 3. Effects of Lactobacillus casei R-1 fermentation product on microbial parameters of toast bread shelf life.

[0086]

[0087] Results analysis:

[0088] As shown in Table 3, with the increase of R-1 whey starter culture addition, the total bacterial count, mold count, and Staphylococcus aureus count of bread during storage all showed a decreasing trend, and were all lower than the blank control group, indicating that it can effectively inhibit microbial growth and extend shelf life. Figure 3-1 As shown, the mold growth in the control group was significantly greater than that in the experimental group, so adding Lactobacillus casei R-1 fermentation product inhibited mold growth.

[0089] like Figure 3-2 As shown, the mold count in the blank control group bread exceeded the safety limit on the 7th day of storage. Although other strains of fermented products had some effect, they were far inferior to those of this invention. Bread with more than 0.8% of the R1 fermented product of this invention can have its shelf life extended to more than 9 days, far exceeding the blank control (7 days) and the chemical preservative group (about 5 days), with a shelf life extension rate of more than 100%.

[0090] Experimental Example 4: Texture Analysis

[0091] 1. Sample preparation: Cut the cooled bread into uniform slices with a thickness of 2.0 cm and place them on the texture analyzer testing platform.

[0092] 2. Test conditions: Two compression tests were conducted using an APN / 25N cylindrical probe. Parameters were set as follows: speed before test 1.0 mm / s, speed during test 1.0 mm / s, speed after test 1.0 mm / s, compression deformation 50%, interval between two compressions 5 seconds, and trigger force 5 g.

[0093] 3. Index Measurement: The instrument automatically records and calculates textural parameters such as hardness, elasticity, cohesiveness, adhesiveness, and chewiness.

[0094] Results analysis: such as Figure 4 As shown, when the addition amount of *Lactobacillus casei* R-1 ferment broth is between 0.4% and 0.6%, the various textural indicators are relatively ideal, enabling the bread to achieve a good balance in terms of hardness, elasticity, adhesiveness, and chewiness. At an addition amount of approximately 0.4% to 0.5%, the hardness is relatively low; at 0.4% to 0.6%, the chewiness is good. Among these, the sample with an addition amount of 0.5% maintains suitable hardness while exhibiting good elasticity and cohesion, moderate adhesiveness and chewiness, resulting in the best overall taste.

[0095] Experimental Example 5: Flavor Analysis

[0096] 1. Sample pretreatment: Accurately weigh 1.0 g of bread crumb sample, tear it into small pieces with sterile forceps and place it in a 20 mL headspace vial, then seal.

[0097] 2. Instrumental analysis: Gas chromatography-ion mobility spectrometry (GC-IMS) was used for analysis.

[0098] (1) Chromatographic conditions: WAXMS polar capillary column, 30 m × 0.32 mm × 0.25 μm; column temperature program: initial 40℃ for 1 min, increase to 100℃ at 2℃ / min and hold for 31 min, then increase to 180℃ at 5℃ / min and hold for 62 min; IMS conditions: high-purity nitrogen as drift gas, flow rate 1.0 mL / min, temperature 45℃. The total scan mass range was 33–495 m / z.

[0099] 3. Data processing: Using the NIST database in the instrument's software, combined with the retention index (RI), spectral analysis, substance identification, and difference comparison were performed on the volatile organic compounds (VOCs) in the samples to evaluate the effect of R-1 whey fermentation product on the flavor compounds of toast bread.

[0100] Scale settings:

[0101] Comparative Example 1 Chemical Preservative Control: 0.1% (w / w) of calcium propionate was added to the bread recipe of Example 2;

[0102] Comparative Example 2 Unfermented Substrate Control: 1.0% (w / w) of unfermented whey protein powder was added to the bread recipe of Example 2;

[0103] Comparative Examples 3-5: Other microbial fermentation products as controls;

[0104] The only difference between Comparative Example 3 and Example 1 is that Lactobacillus plantarum HJ-S2 was used, while the other conditions were the same as in Example 1;

[0105] The only difference between Comparative Example 4 and Example 1 is the presence of Lactobacillus paracasei DS31; all other conditions are the same as in Example 1.

[0106] The only difference between Comparative Example 5 and Example 1 is that Lactobacillus casei R6 was used, while the other conditions were the same as in Example 1;

[0107] Comparative Examples 6-7: Fermentation Parameter Control;

[0108] The only difference between Comparative Example 6 and Example 1 is that the fermentation time is 16 hours, and the other conditions are the same as in Example 1;

[0109] The only difference between Comparative Example 7 and Example 1 is that the fermentation time is 120 hours, while the other conditions are the same as in Example 1;

[0110] Table 4-5 Effects of fermentation products from different strains on the texture and flavor of toast bread

[0111]

[0112] Results analysis:

[0113] (1) Principal Component Analysis (PCA): such as Figure 5 As shown, samples with different addition amounts showed clear separation on principal component 1 (PC1), and the scores of samples with different addition amounts differed significantly on the PC-1 axis; the 0.3% addition amount sample points were more dispersed and showed little difference; the 1% addition amount sample points were more clearly separated from the samples with other addition amounts, and had a significant impact on toast bread. This indicates that the addition of R-1 leavening agent is the main factor leading to differences in bread flavor.

[0114] (2) Flavor substance heatmap:

[0115] according to Figure 6 Flavor compound heatmaps and data analysis revealed characteristic flavor compound profiles in bread at different addition concentrations, which can be summarized into three main regions:

[0116] a. Low addition range (0.3%–0.5%): Within this range, the relative contents of butyraldehyde (grassy aroma), limonene (lemon aroma), phenylacetaldehyde (floral and honey aroma), and 2-methylbutyl acetate (fruity aroma) in the bread were significantly higher than those in other groups and the blank control. These substances together constitute a pleasant flavor profile dominated by fresh fruity and citrus aromas.

[0117] b. Medium addition range (0.5%–0.7%):

[0118] The addition of 0.5% is a key turning point, at which point the flavor composition is the richest and most harmonious. In addition to retaining some fruity aroma characteristics, key flavor compounds such as ethyl 2-methylbutyrate (apple and strawberry aroma), methylthion (potato and tomato aroma), and 2,3-pentanedione (creamy aroma) are significantly enhanced, forming a rich, harmonious, and balanced overall flavor.

[0119] At an addition level of 0.7%, alcohols such as 2-pentanol and cis-2-penten-1-ol exhibit distinctive characteristics, giving the bread a unique and mellow aroma.

[0120] c. High addition range (0.8%–1.0%): In this range, the flavor profile changes significantly, with ketones and esters, represented by 2-heptanone (blue cheese, musty aroma), n-butyl lactate (fruity aroma), p-cymene (citrus, herbaceous aroma), and isoamyl isovalerate (banana aroma), becoming dominant. This makes the bread flavor more inclined towards a rich fermented ester aroma and a slight ketone characteristic.

[0121] The flavor analysis results indicate that the amount of *Lactobacillus casei* R-1 ferment broth added is a key factor in regulating the final flavor characteristics of toast bread. Considering the flavor composition and sensory relevance at various concentrations, an addition of 0.5% can simultaneously inhibit microorganisms while imparting the richest, most harmonious, and pleasant flavor profile to the toast bread, achieving the optimal balance between preservation and flavor enhancement.

[0122] The above embodiments are preferred embodiments of the present invention, but the actual embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for preparing lactic acid bacteria ferment for bread preservation, characterized in that, Includes the following steps: Provided is Lacticaseibacillus casei R-1, with accession number CGMCC NO.33871; The Lactobacillus casei R-1 was inoculated into a culture medium containing whey protein for fermentation to obtain a fermentation broth; The fermentation broth is post-processed to obtain the lactic acid bacteria fermentation product.

2. The method according to claim 1, characterized in that, The fermentation conditions include: The fermentation temperature is 28-40 ℃, preferably 37 ℃; The fermentation time is 16-120 hours, preferably 72 hours; The inoculation amount is 3%-7% (v / v), preferably 5% (v / v).

3. The method according to claim 1 or 2, characterized in that, The post-processing includes: Centrifuge the fermentation broth and collect the supernatant; Optionally, the supernatant may be filtered to remove bacteria; The supernatant was freeze-dried to obtain freeze-dried powder.

4. A lactic acid bacteria fermentation product prepared by the method according to any one of claims 1-3.

5. A type of bread, characterized in that, The bread contains the lactic acid bacteria ferment as described in claim 4.

6. The bread according to claim 5, characterized in that, Based on the total mass of flour (100%), the amount of lactic acid bacteria ferment added is 0.3%-1.0% (w / w), preferably 0.5%-0.8% (w / w).

7. The bread according to claim 5 or 6, characterized in that, The bread in question is toast.

8. Use of the lactic acid bacteria fermentation product according to claim 4 in the preparation of baked goods.

9. The use according to claim 8, characterized in that, The intended use is to inhibit microorganisms in baked goods and / or improve the flavor of baked goods and / or delay the aging of baked goods.

10. The use according to claim 9, characterized in that, The microorganisms include bacteria and / or molds, preferably one or more of Staphylococcus aureus, Escherichia coli and Aspergillus niger.

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