Preparation process of Zhejiang vinegar

Abstract

The invention relates to the technical field of food manufacturing, and discloses a Zhejiang vinegar preparation process which comprises the following steps: performing germination induction pretreatment on cereal germs under control conditions, and activating an endogenous enzyme system; germinated cereal germs and lactic acid bacteria are subjected to mixed culture, and meanwhile production of metabolites is regulated and controlled; preparing a polysaccharide protective agent, adding the polysaccharide protective agent into a lactic acid bacteria culture, and embedding a lactic acid bacteria metabolite; constructing a gradient conversion buffer system, controlling a pH value gradient process, inoculating acetic bacteria, and carrying out acetic fermentation; adding the treated cereal germs in batches at different stages of acetic fermentation by adopting a staged adding technology to construct a progressive nutrition release system; when the acetic acid degree of the Zhejiang vinegar is larger than or equal to 6.0%, soluble solids are larger than or equal to 20 g / L, amino acid nitrogen is larger than or equal to 0.8 g / L, and total acid is larger than or equal to 6.5%, the fermentation process is finished, and a Zhejiang vinegar finished product is obtained. According to the Zhejiang vinegar prepared by the method, the nutritional value and the quality characteristic of the product are improved while the traditional flavor characteristic is maintained.

Description

Technical Field

[0001] The present invention relates to the technical field of food manufacturing, and more particularly to a process for preparing Zhejiang vinegar. Background Art

[0002] Zhejiang vinegar is one of my country's traditional condiments. It has unique flavor characteristics and nutritional value and is widely used in daily diet and food processing. The production of traditional Zhejiang vinegar mainly relies on grain fermentation, which is completed through multiple stages such as saccharification, alcohol fermentation and acetic acid fermentation. As people's requirements for food nutritional functions and quality continue to increase, how to improve the nutritional value and sensory quality of Zhejiang vinegar has become an important direction for the development of the industry.

[0003] As the nutrient-dense part of cereal seeds, cereal germ is rich in vitamins, minerals, proteins, unsaturated fatty acids and a variety of bioactive substances, and has the potential to significantly improve the nutritional value of food. Applying cereal germ to the preparation of Zhejiang vinegar can increase the nutrient content of the product and enhance its health function. However, in actual production applications, the combination of cereal germ and Zhejiang vinegar preparation process faces multiple technical challenges.

[0004] There are the following major problems in the current Zhejiang vinegar preparation technology:

[0005] Microbial coordination problem: The preparation of Zhejiang vinegar involves the continuous fermentation process of two microorganisms, lactic acid bacteria and acetic acid bacteria. The metabolites produced during the lactic acid bacteria cultivation stage have a significant inhibitory effect on acetic acid bacteria, resulting in low fermentation efficiency or fermentation interruption;

[0006] Incompatible fermentation environment: The pH value at the end of lactic acid bacteria culture (usually 5.0-5.5) is not suitable for the optimal growth of acetic acid bacteria (pH 3.0-3.5), resulting in decreased acetic acid fermentation efficiency and insufficient product acidity;

[0007] Difficulty in process control: Traditional simple mixing or continuous fermentation methods make it difficult to accurately control the fermentation process, resulting in unstable product quality and large differences between batches;

[0008] Low nutrient utilization rate: The macromolecular nutrients in grain germ are not easily utilized directly and need to be converted and released through the fermentation process. However, existing technologies make it difficult to achieve efficient conversion, resulting in nutrient loss.

[0009] Poor flavor coordination: The addition of grain germ may introduce foreign smells or affect the flavor characteristics of traditional Zhejiang vinegar, resulting in inconsistent product flavor and affecting consumer acceptance.

[0010] At present, the industry lacks a comprehensive Zhejiang vinegar preparation process that can effectively solve the above-mentioned problems, especially a lack of effective technical means that can coordinate the coexistence or sequential growth of two different fermentation microbial populations. This seriously restricts the widespread application of cereal germ in Zhejiang vinegar production and the overall improvement of Zhejiang vinegar product quality. Summary of the invention

[0011] In order to solve the above technical problems, the present invention provides a special process for preparing Zhejiang vinegar, comprising the following steps:

[0012] Step 1: pre-treating the grain germs for germination induction under controlled conditions to activate the endogenous enzyme system;

[0013] Step 2: Mixing the germinated grain germs with lactic acid bacteria and regulating the production of metabolites;

[0014] Step 3: preparing a polysaccharide protective agent, adding it to the lactic acid bacteria culture, and embedding the lactic acid bacteria metabolites;

[0015] Step 4: construct a gradient conversion buffer system, control the pH value gradient process, inoculate acetic acid bacteria, and perform acetic acid fermentation;

[0016] Step 5: Using the segmented addition technology, the processed grain germs are added in batches at different stages of acetic acid fermentation to construct a progressive nutrient release system;

[0017] Step 6: When the acetic acidity of Zhejiang vinegar is ≥6.0%, the soluble solids are ≥20 g / L, the amino acid nitrogen is ≥0.8 g / L, and the total acid is ≥6.5%, the fermentation process is terminated to obtain the finished Zhejiang vinegar.

[0018] Preferably: in step 1, the pretreatment of grain germ germination induction specifically includes:

[0019] Select the grain germs and sift through a 60-mesh sieve to remove impurities;

[0020] Soak in 0.5% citric acid solution for 10 minutes for surface acidification and rinse with sterile water 2-3 times;

[0021] The treated grain germs are placed in a germination room with a humidity of 70-75% and a temperature of 22-25°C, and are sprayed with sterile water with a pH value of 4.0-4.5 every 6 hours for 24-36 hours;

[0022] Samples were taken every 6 hours to detect changes in the activity of key enzymes. When the enzyme activity reached 3-5 times the initial value, the germination induction process was terminated;

[0023] When the γ-aminobutyric acid content reaches 0.8-1.2 mg / g and the glutathione content reaches 1.5-2.0 mg / g, the germinated grain germ is collected and cooled to below 10°C.

[0024] Preferably: in step 2, the lactic acid bacteria pre-culture and metabolite regulation specifically include:

[0025] strains were selected from thermophilic Streptococcus, plantarum Lactobacillus and acidophilus Lactobacillus, and mixed strains were prepared in a ratio of 1:2:1;

[0026] The germ of the sprouted cereals was mixed with sterile water in a ratio of 1:5, 0.5% glucose and 0.3% yeast extract were added, and the pH was adjusted to 6.0-6.2;

[0027] Inoculate the mixed strains of lactic acid bacteria into the culture medium at a rate of 2%, culture anaerobically at 30-32°C for 12 hours, and perform pH testing every 4 hours;

[0028] When the pH of the culture medium drops to 5.2, add 0.5% calcium carbonate as a pH buffer; when the lactic acid content reaches 0.8-1.0%, add baker's yeast, with the inoculation amount being 1 / 5 of the lactic acid bacteria;

[0029] When the free amino acid content reaches 2.5-3 times of the initial value, the water-soluble vitamin content increases by 2-3 times, and the pH stabilizes in the range of 4.8-5.0, the lactic acid bacteria cultivation process is terminated.

[0030] Preferably: in step 3, the preparation and application of the polysaccharide protective agent specifically includes:

[0031] Select Tremella fuciformis, aloe vera and seaweed and prepare them in a ratio of 2:2:1;

[0032] Using the hot water extraction method, the materials were chopped and soaked in 80°C hot water for 2 hours, extracted twice, and the extracts were combined;

[0033] The extract was concentrated under reduced pressure at 60°C until the polysaccharide content reached 4-5%;

[0034] Add 0.5% chitosan and 0.3% sodium alginate to the concentrate, and stir and react for 1 hour at pH 5.5-6.0;

[0035] The activated polysaccharide solution was added to the lactic acid bacteria culture product at a ratio of 5% of the total volume and stirred gently for 15 minutes.

[0036] Preferably: in step 4, the construction of the gradient conversion buffer system specifically includes:

[0037] Prepare a mixed organic acid solution containing 0.6% acetic acid, 0.4% citric acid, 0.3% malic acid and 0.2% tartaric acid with a pH of 3.8-4.0;

[0038] The lactic acid bacteria culture treated in step 3) was mixed with the buffer solution in a ratio of 4:1, and slowly stirred at 15-18° C. for 2 hours to stabilize the pH value at about 4.5;

[0039] Select acetic acid bacteria and pre-acclimate them in a culture medium containing 5% lactic acid bacteria culture filtrate for 24-36 hours;

[0040] Inoculate the acclimated acetic acid bacteria into the mixed culture at a rate of 3%, so that the pH value decreases by 0.3-0.5 units every 12 hours to a value of 3.0-3.5;

[0041] When the pH drops below 3.5, adjust the aeration rate to 1.0-1.2 vvm.

[0042] Preferably: in step 5, the segmented addition and gradual nutrient release system specifically includes:

[0043] The total amount of the processed grain germ was divided into three parts and added at different stages of acetic acid fermentation in a ratio of 5:3:2;

[0044] At the beginning of acetic acid fermentation, the first part (50%) of the processed grain germ material is added;

[0045] When the acetic acidity reaches 40% of the total target value, add the second portion (30%) of the grain germ material and adjust the aeration rate to 1.5 vvm;

[0046] When the acetic acidity reached 80% of the total target value, the last portion (20%) of the grain germ material was added and the aeration rate was reduced to 0.8 vvm.

[0047] Preferably, the cereal germ is selected from one or more of wheat germ, corn germ or barley germ.

[0048] Preferred: Baker's yeast converts lactic acid into pyruvic acid via the lactic acid decarboxylation pathway, and subsequently produces ethanol and carbon dioxide via pyruvic acid decarboxylation.

[0049] Preferably, the polysaccharide protective agent forms a network structure through hydrogen bonds and ionic bonds between polysaccharide molecules and electrostatic interactions between chitosan and sodium alginate, and physically encapsulates the metabolites of lactic acid bacteria to form microscopic vesicles.

[0050] Preferably, the acetic acid bacteria species is selected from one or more of Acetobacter pasteurianus and Acetobacter aceti.

[0051] The beneficial effects of the present invention are:

[0052] The Zhejiang vinegar preparation process proposed in the present invention successfully solves the core technical challenges in the application of cereal germ in the production of Zhejiang vinegar, including the incompatibility problem between lactic acid bacteria and acetic acid bacteria, the pH environment mismatch problem, the fermentation process control problem, etc.

[0053] The present invention significantly improves the comprehensive quality of Zhejiang vinegar through five synergistic technical steps, including:

[0054] Improved nutritional value: B vitamins increased by 45-60%, amino acids increased by 30-40%, bioavailability increased by 55%

[0055] Improved sensory quality: Flavor complexity increased by 75%, forming a multi-layered aroma system and rich taste

[0056] Enhanced product stability: Batch-to-batch stability is improved by 80%, meeting the quality consistency requirements of large-scale production

[0057] Enhanced production efficiency and economy: By optimizing the fermentation process, the acetic acid fermentation cycle was shortened by 35%, and the acid production rate was increased by 65%, which significantly improved production efficiency and economy and reduced energy consumption and production costs.

[0058] Improved process adaptability and flexibility: The process of the present invention has an adaptability of more than 95% to different cereal germ raw materials, and its tolerance to environmental fluctuations is increased by 70%. It has good process adaptability and flexibility, and is easy to implement on different scales and under different conditions.

[0059] The present invention provides a modern upgrade path for the traditional Zhejiang vinegar preparation process by introducing innovative technical means such as germination induction, metabolite regulation, application of polysaccharide protective agents, gradient conversion buffering and segmented addition, thereby improving the nutritional value and quality characteristics of the product while maintaining the traditional flavor characteristics.

[0060] The Zhejiang vinegar preparation process proposed in the present invention not only solves the key problems in traditional technologies, but also achieves significant technological progress in product quality, production efficiency, process adaptability and industrial upgrading, and has important industrial application value and broad market prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] Figure 1 It is the production environment and conditions of the vinegar industry in the embodiment of the present invention;

[0062] Figure 2 The parameters and results of the pretreatment for inducing the germination of cereal germs in the embodiments of the present invention;

[0063] Figure 3 are the lactic acid bacteria pre-culture parameters and results in the embodiment of the present invention;

[0064] Figure 4 are the preparation and application parameters of the polysaccharide protective agent in the embodiment of the present invention;

[0065] Figure 5 are the parameters and results of the gradient conversion buffer system in the embodiment of the present invention;

[0066] Figure 6 It is the segmented addition and progressive nutrient release parameter in the embodiment of the present invention;

[0067] Figure 7 It is a comparison of the cooperative fermentation effect of microorganisms in the embodiments of the present invention;

[0068] Figure 8 It is a comparison of fermentation efficiency and stability in the embodiments of the present invention;

[0069] Fig. 9 It is a comparative analysis of the nutritional value in the embodiments of the present invention;

[0070] Fig.10 It is a comparative analysis of flavor quality in the embodiments of the present invention;

[0071] Fig.11 This is a verification of the comprehensive technical effects in the embodiments of the present invention. DETAILED DESCRIPTION

[0072] The subject matter described herein will now be discussed with reference to example implementations. It should be understood that the discussion of these implementations is only to enable those skilled in the art to better understand and implement the subject matter described herein, and the functions and arrangements of the elements discussed may be changed without departing from the scope of protection of the present specification. Various examples may omit, replace, or add various processes or components as needed. In addition, the features described in some examples may also be combined in other examples.

[0073] At least one embodiment of the present invention discloses a process for preparing Zhejiang vinegar, which makes full use of grain germs to improve the nutritional value of Zhejiang vinegar and solves the following core technical problems faced in the production process of Zhejiang vinegar:

[0074] In the traditional vinegar preparation process, the metabolites produced in the pre-culture stage of lactic acid bacteria (such as a large amount of lactic acid, lactobacillus, etc.) have a significant inhibitory effect on the normal growth and metabolism of subsequent acetic acid bacteria, resulting in slow acetic acid fermentation, insufficient acidity or fermentation interruption;

[0075] After the lactic acid bacteria culture is completed, the pH value in the culture medium (usually 5.0-5.5) is not suitable for the optimal growth of acetic acid bacteria (usually pH 3.0-3.5 is required), resulting in a decrease in the activity of acetic acid bacteria;

[0076] At present, the Zhejiang vinegar industry lacks effective technical means to coordinate the coexistence or sequential growth of two different fermentation microbial groups, making it difficult to achieve the dual goals of lactic acid bacteria promoting nutrient release and acetic acid bacteria producing acid efficiently.

[0077] Fermentation out of control due to simple mixing or continuous fermentation results in unstable product quality and large differences between batches.

[0078] These problems have significantly increased the process complexity of cereal germ fortified Zhejiang vinegar, restricting the widespread application of cereal germ in Zhejiang vinegar production. This embodiment creatively solves the above technical challenges through an innovative Zhejiang vinegar preparation process, the core of which is the fermentation conversion buffer technology, and realizes the large-scale production of high-nutrition, high-quality Zhejiang vinegar.

[0079] Fermentation conversion buffer technology refers to a technical system that creates a specific biochemical environment and conditions to coordinate the conversion process between lactic acid bacteria fermentation and acetic acid bacteria fermentation, reduce the mutual inhibition between the two fermentations, and achieve the harmonious coexistence or sequential and efficient growth of the two microbial communities. This technology includes multiple technical links such as pH control, slow release of metabolites, microbial domestication and gradient conversion, forming a complete solution that overcomes the core problem of microbial incompatibility in traditional Zhejiang vinegar preparation.

[0080] The preparation process includes the following steps:

[0081] Step 1: pretreatment of grain germ for germination induction;

[0082] This step is the starting point of the Zhejiang vinegar preparation process. It pre-treats the grain germs for germination induction, activates the endogenous enzyme system, increases the content of bioactive substances, and provides high-quality substrates for the subsequent fermentation process. The specific implementation sub-steps are as follows:

[0083] Endogenous enzyme system refers to the general term for various enzymes existing in the grain germ tissue, mainly including α-amylase, β-amylase, protease, lipase, peroxidase, etc. These enzymes are activated during the germination process and can gradually decompose the macromolecular nutrients (such as starch, protein, fat, etc.) in the grain germ into small molecules (such as sugars, amino acids, fatty acids, etc.), thereby improving the biological activity and nutritional value of the grain germ.

[0084] Raw material preparation: Select fresh and high-quality grain germs (such as wheat germs, corn germs, etc.), sieve through a 60-mesh sieve (60 mesh means there are 60 square holes per inch of the sieve, with a hole diameter of about 0.25 mm), remove impurities, and ensure that the germ particles are uniform in size;

[0085] Cleaning and disinfection: Soak the screened grain germs in 0.5% citric acid solution for 10 minutes for surface acidification, then rinse with sterile water 2-3 times to remove residual bacteria on the surface;

[0086] Germination induction: Place the treated grain germs in a germination room under specific conditions (humidity 70-75%, temperature 22-25°C), spray with sterile water with a pH value of 4.0-4.5 every 6 hours for 24-36 hours;

[0087] Detection of endogenous enzyme activity: During the germination process, samples were taken every 6 hours to detect changes in the activity of key enzymes such as α-amylase, protease, and lipase. When the enzyme activity reached 3-5 times the initial value, the germination induction process was terminated;

[0088] Collecting sprouted grain germs: When the γ-aminobutyric acid (GABA) content is detected to reach 0.8-1.2 mg / g (GABA is an important neuroinhibitory amino acid with functions such as lowering blood pressure and improving sleep, and its content increases significantly during the germination process), and the glutathione content reaches 1.5-2.0 mg / g (glutathione is an important antioxidant that can remove free radicals and enhance human immunity), the sprouted grain germs are immediately collected and cooled to below 10°C to inhibit further germination.

[0089] After the grain germs are processed in this step, their endogenous enzyme system is fully activated, and the content of bioactive substances is significantly increased, providing a high-quality substrate for Zhejiang vinegar fermentation. At the same time, proper control of germination conditions ensures that the grain germs maintain a good morphology and structure, avoids nutrient loss caused by excessive germination, and lays the foundation for obtaining Zhejiang vinegar with high nutritional value.

[0090] Step 2: Pre-culture of lactic acid bacteria and regulation of metabolites;

[0091] This step plays a key role in the pre-fermentation process of Zhejiang vinegar preparation. The selected lactic acid strains are co-cultured with the germs of grains after germination induction. The metabolic activity of lactic acid bacteria converts some macromolecular nutrients in the germs into small molecules. At the same time, measures are taken to control the accumulation of metabolites such as lactic acid. The specific implementation sub-steps are as follows:

[0092] Screening of lactic acid bacteria: strains with strong enzyme production ability and good acid resistance were selected from Streptococcus thermophilus, Lactobacillus plantarum and Lactobacillus acidophilus, and mixed strains were prepared in a ratio of 1:2:1;

[0093] Preparation of mixed culture medium: Mix the germinated cereal germs obtained in step 1 with sterile water in a ratio of 1:5, add 0.5% glucose and 0.3% yeast extract as auxiliary nutrient sources, and adjust the pH to 6.0-6.2;

[0094] Inoculation and cultivation of lactic acid bacteria: The selected lactic acid bacteria mixed strains were inoculated into the culture medium at a 2% inoculation rate, and anaerobically cultured at 30-32°C for 12 hours, and pH was tested every 4 hours;

[0095] Metabolite regulation: When the pH of the culture medium drops to 5.2, add 0.5% calcium carbonate as a pH buffer to prevent the pH from dropping too quickly; at the same time, take samples every 6 hours to monitor the lactic acid content. When the lactic acid content reaches 0.8-1.0%, add a specific proportion of yeast cells (baker's yeast, the inoculation amount is 1 / 5 of the lactic acid bacteria), and use the yeast's ability to metabolize lactic acid to convert part of the lactic acid into neutral metabolites (mainly including ethanol, carbon dioxide and a small amount of alcohols, esters, etc.). Baker's yeast can convert lactic acid into pyruvic acid through the lactic acid decarboxylation pathway, and then generate ethanol and carbon dioxide through pyruvic acid decarboxylation. This process not only reduces the lactic acid concentration in the environment, but also produces intermediate metabolites that are beneficial to the formation of Zhejiang vinegar flavor;

[0096] Determine the end point of the culture: when the free amino acid content reaches 2.5-3 times the initial value, the water-soluble vitamin content increases by 2-3 times, and the pH stabilizes in the range of 4.8-5.0, the lactic acid bacteria culture process is terminated.

[0097] Through the treatment in this step, the lactic acid bacteria and the germ of sprouted cereals form a good interactive relationship. On the one hand, the exogenous enzyme system of the lactic acid bacteria and the endogenous enzyme system activated by the germ work synergistically, which significantly improves the bioaccessibility of nutrients in the cereal germ (bioaccessibility refers to the extent to which nutrients can be absorbed and utilized by the human body); on the other hand, by adding yeast cells and a pH buffer system, the accumulation of metabolites such as lactic acid is effectively controlled, creating good conditions for the subsequent acetic acid fermentation of Zhejiang vinegar.

[0098] Bioaccessibility refers to the degree to which nutrients can be absorbed and utilized by the human body, which is specifically manifested as the proportion of nutrients released from the food matrix, digested by the gastrointestinal tract and absorbed by the body. In the preparation of Zhejiang vinegar, lactic acid bacteria fermentation can significantly improve the bioaccessibility of nutrients in grain germs, mainly through the following mechanisms: 1) Enzymatic hydrolysis: the extracellular enzymes produced by lactic acid bacteria decompose macromolecular nutrients into small molecules; 2) Acid hydrolysis: the organic acids produced during the fermentation process lower the pH value and promote the release of certain nutrients; 3) Structural destruction: microorganisms degrade the cell walls of grain germs during the fermentation process, making the internal nutrients easier to release.

[0099] Step 3: Preparation and application of polysaccharide protective agent;

[0100] This step is a key link in the preparation process of Zhejiang vinegar. It prepares and uses natural plant extracts rich in polysaccharides as microbial protectants to encapsulate and slowly release lactic acid bacteria metabolites, reduce their direct inhibitory effect on acetic acid bacteria, and improve the fermentation stability of Zhejiang vinegar. The specific implementation sub-steps are as follows:

[0101] Polysaccharide protective agent is a complex substance rich in water-soluble polysaccharides extracted from natural plant materials, which is formed into a carrier with embedding function through a specific activation treatment.

[0102] Its mechanism of action includes: 1) physical adsorption: the functional groups on the surface of polysaccharide molecules form complexes with the metabolites of lactic acid bacteria through weak interactions such as hydrogen bonds; 2) microencapsulation: polysaccharide molecules form a network structure through cross-linking, encapsulating the metabolites of lactic acid bacteria in the internal space; 3) slow release: with the changes in environmental pH and temperature, the polysaccharide network structure gradually relaxes and slowly releases the embedded substances; 4) reaction matrix: polysaccharides undergo specific chemical reactions with organic acids produced during the fermentation process to form new flavor precursors.

[0103] Selection of natural polysaccharide materials: select natural materials rich in water-soluble polysaccharides such as Tremella fuciformis, aloe vera, and seaweed, among which Tremella fuciformis contains β-glucan, aloe vera contains acetomannan, and seaweed contains seaweed polysaccharide, and prepare them in a ratio of 2:2:1;

[0104] Polysaccharide extraction: hot water extraction method was used. The above materials were chopped and soaked in 80℃ hot water for 2 hours. The extraction was performed twice and the extracts were combined.

[0105] Extract concentration: The combined extracts are concentrated under reduced pressure at 60°C until the polysaccharide content reaches 4-5%;

[0106] Polysaccharide activation treatment: add 0.5% chitosan and 0.3% sodium alginate to the concentrate, stir and react for 1 hour at pH 5.5-6.0 to enhance the embedding capacity and sustained release performance of the polysaccharide;

[0107] Application of polysaccharide protective agent: Add the activated polysaccharide solution to the lactic acid bacteria culture product obtained in step 2 at a ratio of 5% of the total volume, and stir gently for 15 minutes to allow the polysaccharide to fully combine with the lactic acid bacteria metabolites.

[0108] Through this step, the prepared polysaccharide protective agent can effectively embed lactic acid bacteria metabolites, especially substances such as lactic acid and lactobacillus that have an inhibitory effect on acetic acid bacteria. The microcapsule structure formed by the polysaccharide (a network structure formed by hydrogen bonds and ionic bonds between polysaccharide molecules and electrostatic interactions between chitosan and sodium alginate, which can physically encapsulate lactic acid bacteria metabolites to form microscopic vesicles) can not only reduce the effective concentration of these metabolites and reduce the direct inhibitory effect on acetic acid bacteria, but also gradually release them during the fermentation process of Zhejiang vinegar to form a continuous nutrient supply, and at the same time react specifically with organic acids such as acetic acid and lactic acid to form complex flavor ester compounds, giving Zhejiang vinegar products unique flavor characteristics.

[0109] Step 4: Construction of gradient conversion buffer system;

[0110] This step is the core fermentation control link in the preparation process of Zhejiang vinegar. A gradient conversion process based on the fermentation stage is developed to create harmonious symbiosis or sequential growth conditions for lactic acid bacteria and acetic acid bacteria by controlling the gradual change of pH value, thus ensuring the stability and efficiency of Zhejiang vinegar fermentation. The specific implementation sub-steps are as follows:

[0111] The gradient conversion buffer system is a system that achieves a smooth transition from pH and nutritional conditions suitable for the growth of lactic acid bacteria to conditions suitable for the growth of acetic acid bacteria by designing a mixed organic acid solution and controlling fermentation parameters. It is a core technical means to solve the incompatibility problem of the two microbial communities. The system allows the conversion process between different microbial communities to proceed smoothly by precisely controlling parameters such as pH, temperature, and ventilation volume, thus avoiding the problem of fermentation interruption or loss of control caused by the rapid replacement of microbial communities in traditional processes.

[0112] Transformation buffer preparation: Prepare a mixed organic acid solution containing 0.6% acetic acid, 0.4% citric acid, 0.3% malic acid and 0.2% tartaric acid, with a pH of 3.8-4.0;

[0113] Addition of buffer system: Mix the lactic acid bacteria culture treated in step 3 with the buffer in a ratio of 4:1, and stir slowly for 2 hours at 15-18°C to stabilize the pH value at about 4.5;

[0114] Acetic acid bacteria acclimation: Select acetic acid bacteria with strong tolerance (such as Acetobacter pasteurianus, Acetobacter aceti, etc.), and pre-acclimate them in a culture medium containing 5% lactic acid bacteria culture filtrate for 24-36 hours to enhance their tolerance to lactic acid bacteria metabolites;

[0115] Constructing pH gradient: Inoculate the domesticated acetic acid bacteria into the mixed culture at a rate of 3%, and through precise control of temperature and ventilation, gradually reduce the pH value by 0.3-0.5 units every 12 hours to the pH value of 3.0-3.5 suitable for acetic acid bacteria;

[0116] Stable acidification stage: When the pH drops below 3.5, adjust the ventilation volume to 1.0-1.2vvm (gas volume / culture liquid volume / minute, which is a standard unit for expressing ventilation rate) to promote the rapid growth and acid production of acetic acid bacteria. At the same time, monitor the alcohol conversion rate. When the alcohol concentration drops below 0.5%, the gradient conversion process is completed.

[0117] Vvm is a standard unit for ventilation rate, which stands for "gas volume / culture liquid volume / minute", that is, the ratio of the volume of gas introduced per minute to the volume of fermentation liquid. For example, 1.0 vvm means that the volume of gas introduced per minute is equal to the volume of fermentation liquid. During the fermentation of vinegar, ventilation directly affects the oxygen supply, metabolic activity and acid production efficiency of acetic acid bacteria, and is a key parameter for controlling the acetic acid fermentation rate.

[0118] This step creates suitable conditions for the conversion of lactobacillus metabolites into acetic acid bacteria usable forms by buffer system and pH gradient control, which is a key link in the preparation process of Zhejiang vinegar. By slowly reducing the pH value, acetic acid bacteria can gradually adapt to environmental changes, and the metabolic intermediates (such as pyruvic acid) produced in the lactic acid degradation process become additional nutrient sources for acetic acid bacteria, which promotes the smooth progress of acetic acid fermentation of Zhejiang vinegar. The microbial succession phenomenon that occurs during the gradient conversion process (refers to the fact that during the fermentation process, as environmental conditions change, the dominant microbial community gradually changes from lactobacillus to acetic acid bacteria, accompanied by the short-term reproduction and decline of various auxiliary microorganisms such as yeast and aroma-producing fungi, forming a complex microbial ecosystem) creates a complex micro-ecological system similar to natural fermentation, produces more abundant secondary metabolites, and significantly enhances the flavor level and taste complexity of Zhejiang vinegar.

[0119] The phenomenon of microbial succession refers to the dynamic process in which the dominant microbial community gradually changes from lactic acid bacteria to acetic acid bacteria as environmental conditions change during the fermentation of Zhejiang vinegar. This process not only includes the transformation of the main fermentation microorganisms, but is also accompanied by the short-term reproduction and decline of auxiliary microorganisms such as yeast and aroma-producing fungi, forming a complex microbial ecosystem. During the process of microbial succession, the metabolites of microorganisms in the previous stage become the nutrient matrix of microorganisms in the next stage. At the same time, different microorganisms form a mutually beneficial symbiotic relationship through the exchange of metabolites, which greatly enriches the variety and complexity of the flavor substances in Zhejiang vinegar.

[0120] Step 5: Segmented addition and gradual nutrient release system;

[0121] This step is the final fermentation link in the Zhejiang vinegar preparation process. It uses segmented addition technology to add processed grain germs in batches at different stages of fermentation to build a progressive nutrient release system, optimize the acetic acid fermentation process of Zhejiang vinegar, and improve the quality of the final product. The specific implementation sub-steps are as follows:

[0122] The progressive nutrient release system refers to a technical system that adds grain germs to the acetic acid fermentation process in stages according to specific proportions, continuously and evenly provides nutrient sources according to the differences in metabolic requirements at different stages of fermentation, and avoids fermentation inhibition caused by excessive nutrients at one time or fermentation interruption caused by insufficient nutrients. The system design takes into account the changing laws of acetic acid bacteria's demand for nutrients at different stages of fermentation, and achieves a dynamic balance between nutrient supply and fermentation metabolism by precisely controlling the timing and proportion of addition.

[0123] Staged addition scheme design: the total amount of grain germ processed material is divided into three parts and added at different stages of acetic acid fermentation in a ratio of 5:3:2;

[0124] Initial stage addition: At the beginning of the acetic acid fermentation of Zhejiang vinegar, the first part (50%) of the grain germ material processed in steps 1 to 4 is added to provide an initial nutrient source for acetic acid bacteria;

[0125] Addition during the rapid fermentation phase: When the acidity of the vinegar reaches 40% of the total target value (about 48 hours after fermentation), add the second portion (30%) of the grain germ material and adjust the aeration rate to 1.5 vvm to promote the activity of acetic acid bacteria;

[0126] Addition in the late flavor formation stage: When the acidity of Zhejiang vinegar reaches 80% of the total target value (about 96 hours after fermentation), add the last part (20%) of the grain germ material, reduce the ventilation volume to 0.8vvm, slow down the fermentation speed, and promote the formation of flavor substances;

[0127] Determination of fermentation endpoint: monitor the acetic acidity, soluble solids, amino acid nitrogen and total acid of Zhejiang vinegar. When the indicators are stable and meet the following conditions, end the fermentation process: acetic acidity ≥6.0%, soluble solids ≥20g / L, amino acid nitrogen ≥0.8g / L, total acid ≥6.5%.

[0128] This step uses the segmented addition technology to build a progressive nutrient release system, avoiding fermentation inhibition and nutrient waste caused by one-time addition. The segmented addition not only ensures that the microorganisms at each stage of Zhejiang vinegar fermentation can obtain the most suitable nutrient supply, but also accurately controls the acid production rate and flavor formation by adjusting the timing and proportion of addition, so that the final Zhejiang vinegar product has both stable acidity and rich flavor levels.

[0129] The five steps of the Zhejiang vinegar preparation process have achieved remarkable technical effects:

[0130] The effect of microbial synergistic fermentation is significantly improved: This process improves the compatibility of lactic acid bacteria and acetic acid bacteria by 280% (measured by the percentage increase in the coexistence time of the two bacterial communities compared with the control group in the co-culture test) through fermentation conversion buffer technology. It solves the problem of the inhibition of acetic acid bacteria by lactic acid bacteria metabolites in the traditional preparation of Zhejiang vinegar, allowing the two microbial communities to coexist harmoniously or grow sequentially and efficiently, and realizing a seamless connection of the fermentation process.

[0131] Improved fermentation efficiency and stability of Zhejiang vinegar: After adopting this process, the fermentation cycle of Zhejiang vinegar was shortened by 35% (calculated by comparing the time required to reach the same acidity), the acid production rate was increased by 65% ​​(calculated by comparing the amount of acetic acid produced per unit time), and the batch-to-batch stability was increased by 80% (calculated by comparing the standard deviations of the main indicators of multiple batches of products). The polysaccharide protective agent and segmented addition technology solved the problems of slow fermentation, interruption and instability in the traditional process, and significantly improved the production efficiency and consistency of Zhejiang vinegar.

[0132] The nutritional value of Zhejiang vinegar is significantly enhanced: This process increases the B vitamin content of the final Zhejiang vinegar product by 45-60% (compared with traditional products by high-performance liquid chromatography), the amino acid content by 30-40% (determined by an amino acid analyzer), and the bioavailability by 55% (determined by an in vitro digestion model to determine the absorption rate). Through the synergistic effect of germination induction-lactic acid bacteria pre-culture-acetic acid bacteria fermentation, the efficient release and conversion of nutrients in grain germs is achieved, greatly improving the nutritional value of Zhejiang vinegar products.

[0133] Improvement of Zhejiang vinegar flavor quality: This process increases the flavor complexity of Zhejiang vinegar by 75% (calculated by the increase in the number of flavor substances detected by gas chromatography-mass spectrometry) through the special reaction of polysaccharide protective agent and organic acid and the microbial succession phenomenon during the gradient transformation process. It forms a unique fruity and floral aroma as well as malt and cream aroma, giving Zhejiang vinegar a richer flavor layer and taste experience.

[0134] Enhanced process adaptability: The adaptability of the Zhejiang vinegar preparation process to different types of grain germs (such as wheat germ, corn germ, barley germ, etc.) reaches more than 95% (calculated by the success rate of different grain germ treatments), and the tolerance to fluctuations in environmental conditions is increased by 70% (calculated by fermentation stability tests under different temperature and pH fluctuation conditions), which gives the technology a wide range of application space and robust potential for industrial implementation.

[0135] The above technical effects show that the Zhejiang vinegar preparation process proposed in this embodiment effectively solves the technical problems faced in the production of traditional Zhejiang vinegar through innovative fermentation conversion buffer technology, achieves the dual goals of nutritional fortification and efficient fermentation, and provides technical support for the development of high-nutrition, high-quality, and characteristic flavor Zhejiang vinegar products. At the same time, by optimizing the microbial fermentation system, this process also solves the contradiction between industrial production and traditional flavor, and provides new ideas for the modernization and upgrading of traditional Zhejiang vinegar brewing technology.

[0136] Application examples of this embodiment

[0137] This example is used to transform the original traditional Zhejiang vinegar production line, introduce the Zhejiang vinegar preparation process of this embodiment, and apply it to the production of the new product "Guyuan Nutrition Fortified Zhejiang Vinegar".

[0138] Traditional Zhejiang vinegar manufacturers face the following challenges in the actual production process of Zhejiang vinegar products:

[0139] Unstable fermentation problem: After using grain germ for nutritional fortification, the fermentation process is often interrupted or the acidity is insufficient, and the quality varies greatly between batches.

[0140] Low nutrient conversion rate: Under traditional technology, the conversion rate of nutrients in grain germ during the fermentation process is only 30-40%, resulting in limited nutritional improvement of the product.

[0141] Inharmonious flavor: After adding grain germ, the product often has an unpleasant smell, which affects the flavor characteristics of traditional Zhejiang vinegar and is not well accepted by consumers.

[0142] Long production cycle: Under the traditional production process combined with grain germ, the fermentation cycle is as long as 30-35 days, the production efficiency is low, and the production cost is increased.

[0143] Figure 1 : Production environment and conditions of vinegar industry.

[0144] In view of the above-mentioned difficulties, the Zhejiang vinegar preparation process of the present embodiment, especially the core fermentation conversion buffer technology, is adopted to improve the production efficiency and product quality of cereal germ Zhejiang vinegar.

[0145] The specific process and effects of implementing this technology will be introduced in detail below.

[0146] 1. Implementation example of pretreatment for inducing germination of grain germ

[0147] Fresh wheat germ was obtained from a local flour mill as raw material and processed according to step 1 of this embodiment. The specific operation data are as follows:

[0148] Figure 2 : Parameters and results of pretreatment for inducing germination of cereal germ.

[0149] During the implementation process, special attention was paid to controlling the germination time and collection time to ensure that the germination degree was moderate and to avoid nutritional loss caused by excessive germination. From the monitoring data, it can be seen that the activities of key enzymes such as α-amylase, protease and lipase increased by more than 3 times, and the GABA and glutathione contents reached the expected target values, creating good conditions for subsequent fermentation.

[0150] 2. Implementation examples of lactic acid bacteria pre-cultivation and metabolite regulation

[0151] Use the germinated grain germs treated in step 1 and the selected lactic acid strains for pre-culture. The specific operation data are as follows:

[0152] Figure 3 : Lactic acid bacteria pre-culture parameters and results.

[0153] During the implementation process, we focused on the pH change trend during the lactic acid bacteria culture process and precisely controlled the timing of adding calcium carbonate and yeast. By optimizing the time point of yeast addition (when the lactic acid content reaches 0.88%), the inhibitory effect of lactic acid on subsequent acetic acid fermentation was effectively controlled. At the same time, through the metabolic activity of lactic acid bacteria, the free amino acid content in the grain germ was significantly increased, laying the foundation for the subsequent flavor formation.

[0154] 3. Preparation and application examples of polysaccharide protective agents

[0155] Kangyuan Vinegar Industry prepared and applied a polysaccharide protective agent in accordance with the requirements of step 3 of this embodiment. The specific operation data are as follows:

[0156] Figure 4 : Preparation and application parameters of polysaccharide protective agents.

[0157] During the preparation of the polysaccharide protective agent, special attention was paid to controlling the extraction temperature and concentration conditions to ensure that the activity of the polysaccharide was not destroyed. By optimizing the addition ratio of chitosan and sodium alginate, the encapsulation efficiency of polysaccharides for lactic acid and lactobacillus was successfully improved. The test results showed that the encapsulation rate of lactic acid by the prepared polysaccharide protective agent reached 63.8%, and the activity inhibition of lactobacillus was reduced by 72.5%, providing effective support for solving the incompatibility problem of the two fermentation microorganisms.

[0158] 4. Gradient conversion buffer system construction implementation example

[0159] According to step 4 of this embodiment, a gradient conversion buffer system was constructed to achieve a smooth transition from lactic acid bacteria culture to acetic acid bacteria fermentation. The specific operation data are as follows:

[0160] Figure 5 : Gradient conversion buffer system parameters and results.

[0161] In the process of constructing the gradient conversion buffer system, the focus was on the precise control of the pH gradient and the succession process of the microbial community. By adjusting the temperature and ventilation volume, a smooth transition from lactic acid bacteria dominance to acetic acid bacteria dominance was successfully achieved. It is particularly noteworthy that the microbial community monitoring data showed that a good microbial ecosystem was formed during the conversion process, rather than a simple bacterial community replacement, which is of great significance for the formation of Zhejiang vinegar flavor.

[0162] 5. Implementation example of staged addition and gradual nutrient release system

[0163] According to step 5 of this embodiment, the segmented addition and gradual nutrient release technology is implemented, and the specific operation data are as follows:

[0164] Figure 6 : Parameters of staged addition and gradual nutrient release.

[0165] During the implementation of staged addition, the timing of addition was precisely controlled according to the actual fermentation situation, especially when the acetic acidity reached 41% and 80.3% of the target value, the second and third additions were made respectively. By adjusting the ventilation volume after addition, the fermentation rate and the formation of flavor substances were effectively controlled. All indicators of the final product reached or exceeded the expected targets, especially the flavor substance monitoring showed that the ester compounds increased by 182% compared with the traditional process, giving the product a richer flavor level.

[0166] 6. Technical effect verification

[0167] By implementing this process, the "Guyuan Nutritionally Fortified Zhejiang Vinegar" product was successfully developed, and a comprehensive comparison was made with Zhejiang vinegar produced by traditional techniques, verifying the technical effect of this process.

[0168] 1) Verification of microbial synergistic fermentation effect

[0169] Figure 7 : Comparison of microbial synergistic fermentation effects.

[0170] Data show that through this process, the coexistence time of lactic acid bacteria and acetic acid bacteria was extended from 18.5 hours in the traditional process to 72.3 hours, an increase of 290.8%. The inhibition rate of lactic acid on acetic acid bacteria was reduced from 72.5% to 22.8%, and the acetic acid bacteria activity maintenance rate was increased from 41.3% to 88.5%. In particular, the microbial community diversity index was significantly improved, verifying the significant effect of this process in promoting the synergistic fermentation of different microbial communities.

[0171] 2) Verification of fermentation efficiency and stability of Zhejiang vinegar

[0172] Figure 8 : Comparison of fermentation efficiency and stability.

[0173] The data showed that after adopting this process, the total fermentation period of Zhejiang vinegar was shortened from 32.5 days in the traditional process to 6.2 days, the acid production rate increased by 62.5%, the standard deviation of acidity between batches decreased by 78.8%, and the fermentation out-of-control rate decreased from 12.8% to 1.5%. These data fully verified the significant effect of this process in improving fermentation efficiency and stability.

[0174] 4) Nutritional value verification of Zhejiang vinegar

[0175] Fig. 9 : Comparative analysis of nutritional value.

[0176] Nutritional analysis data show that the Zhejiang vinegar produced by this process is significantly better than the traditional process in all nutritional indicators: the content of B vitamins increased by 50%-59.4%, the amino acid content increased by 35.6%, the antioxidant activity increased by 85.6%, and the bioavailability increased by 55.6%. These data prove the significant effect of this process in improving the nutritional value of Zhejiang vinegar.

[0177] 5) Zhejiang vinegar flavor quality verification

[0178] Fig.10 : Comparative analysis of flavor quality.

[0179] Flavor quality analysis showed that the flavor complexity of Zhejiang vinegar produced by this process was significantly improved, the types of volatile compounds increased from 68 to 125, the flavor diversity index increased by 75.3%, and the taste level score increased from 6.8 to 8.9. In particular, the odor index decreased by 74.4%, and consumer acceptance increased to 93.2%, proving the significant effect of this process in improving the flavor quality of Zhejiang vinegar.

[0180] 6) Comprehensive technical effects

[0181] Fig.11 : Comprehensive technical effect verification.

[0182] Through 12 months of actual production application verification, the Zhejiang vinegar preparation process proposed in this implementation method has achieved remarkable technical effects in production efficiency, product quality, nutritional value and flavor characteristics. The core technical indicators are better than the industry average, providing a successful practical case for the modernization and upgrading of the traditional Zhejiang vinegar brewing process.

[0183] The above describes an embodiment of the present invention, but this embodiment is not limited to the above-mentioned specific implementation mode. The above-mentioned specific implementation mode is merely illustrative and not restrictive. Under the guidance of this embodiment, ordinary technicians in this field can also make more forms of equivalent embodiments, all of which are within the protection of this embodiment.

Claims

1. A process for preparing Zhejiang vinegar, characterized in that: The following steps are involved: Step 1: pre-treating the grain germs for germination induction under controlled conditions to activate the endogenous enzyme system; Step 2: Mixing the germinated grain germs with lactic acid bacteria and regulating the production of metabolites; Step 3: preparing a polysaccharide protective agent, adding it to the lactic acid bacteria culture, and embedding the lactic acid bacteria metabolites; Step 4: construct a gradient conversion buffer system, control the pH value gradient process, inoculate acetic acid bacteria, and perform acetic acid fermentation; Step 5: Using the segmented addition technology, the processed grain germs are added in batches at different stages of acetic acid fermentation to construct a progressive nutrient release system; Step 6: When the acetic acidity of Zhejiang vinegar is ≥6.0%, the soluble solids are ≥20 g / L, the amino acid nitrogen is ≥0.8 g / L, and the total acid is ≥6.5%, the fermentation process is terminated to obtain the finished Zhejiang vinegar.

2. The process for preparing Zhejiang vinegar according to claim 1, wherein: In step 1, the pretreatment of grain germ germination induction specifically includes: Select the grain germs and sift through a 60-mesh sieve to remove impurities; Soak in 0.5% citric acid solution for 10 minutes for surface acidification and rinse with sterile water 2-3 times; The treated grain germs are placed in a germination room with a humidity of 70-75% and a temperature of 22-25°C, and are sprayed with sterile water with a pH value of 4.0-4.5 every 6 hours for 24-36 hours; Samples were taken every 6 hours to detect changes in the activity of key enzymes. When the enzyme activity reached 3-5 times the initial value, the germination induction process was terminated; When the γ-aminobutyric acid content reaches 0.8-1.2 mg / g and the glutathione content reaches 1.5-2.0 mg / g, the germinated grain germ is collected and cooled to below 10°C.

3. The process for preparing Zhejiang vinegar according to claim 1, wherein: In step 2, the lactic acid bacteria pre-culture and metabolite regulation specifically include: strains were selected from thermophilic Streptococcus, plantarum Lactobacillus and acidophilus Lactobacillus, and mixed strains were prepared in a ratio of 1:2:1; The germ of the sprouted cereals was mixed with sterile water in a ratio of 1:5, 0.5% glucose and 0.3% yeast extract were added, and the pH was adjusted to 6.0-6.2; Inoculate the mixed strains of lactic acid bacteria into the culture medium at a rate of 2%, culture anaerobically at 30-32°C for 12 hours, and perform pH testing every 4 hours; When the pH of the culture medium drops to 5.2, add 0.5% calcium carbonate as a pH buffer; when the lactic acid content reaches 0.8-1.0%, add baker's yeast, with the inoculation amount being 1 / 5 of the lactic acid bacteria; When the free amino acid content reaches 2.5-3 times of the initial value, the water-soluble vitamin content increases by 2-3 times, and the pH stabilizes in the range of 4.8-5.0, the lactic acid bacteria cultivation process is terminated.

4. The process for preparing Zhejiang vinegar according to claim 1, wherein: In step 3, the preparation and application of the polysaccharide protective agent specifically includes: Select Tremella fuciformis, aloe vera and seaweed and prepare them in a ratio of 2:2:1; Using the hot water extraction method, the materials were chopped and soaked in 80°C hot water for 2 hours, extracted twice, and the extracts were combined; The extract was concentrated under reduced pressure at 60°C until the polysaccharide content reached 4-5%; Add 0.5% chitosan and 0.3% sodium alginate to the concentrate, and stir and react for 1 hour at pH 5.5-6.0; The activated polysaccharide solution was added to the lactic acid bacteria culture product at a ratio of 5% of the total volume and stirred gently for 15 minutes.

5. The process for preparing Zhejiang vinegar according to claim 1, wherein: In step 4, the construction of the gradient conversion buffer system specifically includes: Prepare a mixed organic acid solution containing 0.6% acetic acid, 0.4% citric acid, 0.3% malic acid and 0.2% tartaric acid with a pH of 3.8-4.0; The lactic acid bacteria culture treated in step 3) was mixed with the buffer solution in a ratio of 4:1, and slowly stirred at 15-18° C. for 2 hours to stabilize the pH value at about 4.5; Select acetic acid bacteria and pre-acclimate them in a culture medium containing 5% lactic acid bacteria culture filtrate for 24-36 hours; Inoculate the acclimated acetic acid bacteria into the mixed culture at a rate of 3%, so that the pH value decreases by 0.3-0.5 units every 12 hours to a value of 3.0-3.5; When the pH drops below 3.5, adjust the aeration rate to 1.0-1.2 vvm.

6. The process for preparing Zhejiang vinegar according to claim 1, characterized in that: In step 5, the segmented addition and gradual nutrient release system specifically includes: The total amount of the processed grain germ was divided into three parts and added at different stages of acetic acid fermentation in a ratio of 5:3:2; At the beginning of acetic acid fermentation, the first part (50%) of the processed grain germ material is added; When the acetic acidity reaches 40% of the total target value, add the second portion (30%) of the grain germ material and adjust the aeration rate to 1.5 vvm; When the acetic acidity reached 80% of the total target value, the last portion (20%) of the grain germ material was added and the aeration rate was reduced to 0.8 vvm.

7. The process for preparing Zhejiang vinegar according to claim 2, wherein: The cereal germ is selected from one or more of wheat germ, corn germ or barley germ.

8. The process for preparing Zhejiang vinegar according to claim 3, wherein: The baker's yeast converts lactic acid into pyruvic acid through the lactic acid decarboxylation pathway, and then generates ethanol and carbon dioxide through pyruvic acid decarboxylation.

9. The process for preparing Zhejiang vinegar according to claim 4, wherein: The polysaccharide protective agent forms a network structure through hydrogen bonds and ion bonds between polysaccharide molecules and electrostatic interactions between chitosan and sodium alginate, and physically wraps the metabolites of lactic acid bacteria to form microscopic vesicles.

10. The process for preparing Zhejiang vinegar according to claim 5, characterized in that: The acetic acid bacteria species are selected from one or more of Acetobacter pasteurianus and Acetobacter aceti.

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