Preparation method of fermented wind duck with low nitrite residue

Abstract

This invention discloses a method for producing fermented duck with low nitrite residue, characterized by the following steps: marinating duck meat in 1100-1200 mL of marinade per kilogram of duck meat, ensuring the duck embryo is completely submerged in the liquid, for 20-28 hours at a temperature of 0-4°C; then ultrasonically cleaning the duck meat at 150-400W for 1-4 minutes, followed by air drying; and then uniformly spraying a fermentation agent composed of *Lactobacillus fermentum* and *Hansophilus d'Barry* onto the surface of the duck embryo at an inoculation amount of 10... 6 ~10 8 CFU / g, constant temperature and humidity chamber program temperature 14~18℃, relative humidity 65~70%, air drying for 6~8 days, to obtain fermented duck with low nitrite residue. The advantages are that it can better maintain the color of duck meat, efficiently degrade nitrite, inhibit fat oxidation and rancidity, delay the increase of TVB-N value, and extend the product shelf life.

Description

Technical Field

[0001] This invention relates to the technical field of fermented duck, and in particular to a method for producing fermented duck with low nitrite residue. Background Technology

[0002] Duck meat is inherently cool in nature and sweet in taste, with a delicious flavor and rich nutritional value. The edible portion of duck meat contains approximately 16%-25% protein, higher than other livestock meats, and has a moderate and evenly distributed fat content, making it popular among consumers. Air-dried duck meat overcomes the shortcomings of raw meat, such as high moisture content, bland taste, and tender texture, making it a low-fat, high-protein, nutritious meat product with a unique cured flavor. Nitrites are intermediate products in the nitrogen cycle in nature; they can be oxidized to nitrates or reduced to nitrogen gas. Similar in appearance and taste to table salt, they are widely found in vegetables and cured meat products. Nitrites are an important food additive in meat products, possessing color-enhancing, antibacterial, and antioxidant properties. During meat processing, nitrites react with myoglobin to form nitrosomyoglobin, giving meat products a bright red color. Nitrites also effectively inhibit the growth of Clostridium botulinum and Staphylococcus aureus, improving the safety of meat products. However, excessive intake of nitrites seriously harms human health. It has been reported that nitrites undergo nitrosation with secondary amines under acidic conditions in the stomach, leading to the formation of carcinogenic N-nitroso compounds that can cause methemoglobinemia and acute poisoning. Therefore, establishing safe and effective strategies for nitrite degradation is urgently needed.

[0003] Lactic acid bacteria (LAB) and yeast are widely considered safe microorganisms and are extensively used in fermented foods. Numerous studies have demonstrated that they can not only degrade nitrites in food but also enhance flavor and nutritional value. Exploring the interaction patterns between LAB and yeast is beneficial for improving the productivity, sensory properties, and probiotic functions of fermented products. Microbial interaction mechanisms are relatively complex, with metabolic complementarity being the earliest and most widely accepted. LAB metabolites, such as galactose, pyruvate, propionate, and succinate, can promote yeast growth, while yeast produces vitamins and amino acids, or can break down complex carbohydrates into simpler sugars, which is crucial for LAB growth. Utilizing physical fields to reduce nitrites in meat products is a novel and effective method, such as ultrasonic treatment, high-pressure treatment, radiation treatment, electromagnetic radiation, and light treatment. Ultrasonic waves have strong penetrating power and low energy consumption, offering numerous advantages as a novel food processing technology, including high efficiency, safety, environmental friendliness, and low cost. Currently, there are no publicly available research reports, either domestically or internationally, on the application of ultrasonic treatment combined with metabolic complementarity between LAB and yeast in the degradation of nitrites in dried duck meat. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a method for producing fermented duck with low nitrite residue that can better maintain the color of duck meat, efficiently degrade nitrite, inhibit fat oxidation and rancidity, delay the increase of TVB-N value, and extend the shelf life of the product.

[0005] The technical solution adopted by this invention to solve the above-mentioned technical problem is as follows: a method for producing fermented duck with low nitrite residue, comprising the following steps: Washing and draining duck meat is marinated in 1100-1200 mL of marinating liquid per kilogram of duck meat, with the duck embryo completely submerged in the liquid during marinating at a temperature of 0-4°C for 20-28 hours; after marinating, the duck meat is ultrasonically cleaned using an ultrasonic cleaner at a power of 150-400W for 1-4 minutes, then dried; a fermentation agent composed of *Lactobacillus fermentum* and *Hansophilus d'Barry* is evenly sprayed onto the surface of the duck embryo at an inoculation amount of 10... 6 ~10 8 CFU / g, placed in a constant temperature and humidity chamber at a temperature of 14-18℃ and a relative humidity of 65-70%, and air-dried for 6-8 days, yields fermented duck with low nitrite residue.

[0006] Furthermore, the pickling solution is a mixture of 8 wt% sodium chloride and 0.01 wt% sodium nitrite.

[0007] Furthermore, the preparation method of the fermentation agent is as follows: the activated Lactobacillus fermentum and Saccharomyces hansonii were washed three times with sterile physiological saline, and the cell concentration OD was adjusted. 600 =1.0, then mixed by volume ratio of 1:1.

[0008] Furthermore, the Lactobacillus fermentum strain is Lactobacillus fermentum PDD-4, which was deposited at the China General Microbiological Culture Collection Center on March 25, 2019, with accession number CGMCC No. 17434.

[0009] Preferably, the ultrasonic processing power is 250W and the ultrasonic time is 4min.

[0010] Compared with existing technologies, the advantages of this invention are as follows: This invention provides a method for producing fermented duck with low nitrite residue. A nitrite-reducing lactic acid bacteria strain was screened from 12 strains and co-fermented with *Hansøe Bary yeast*, which significantly improves the nitrite degradation rate. *Hansøe Bary yeast* promotes the growth of *Lactobacillus fermentum*, the production of organic and inorganic acids, and the formation of biofilms. The application of ultrasonic treatment combined with co-fermentation of *Lactobacillus fermentum* and *Hansøe Bary yeast* in dried duck meat demonstrates a strong nitrite degradation capacity, with a nitrite degradation rate of 84.55%. This method, when applied to dried duck meat, better preserves the color of the duck meat, efficiently degrades nitrite, inhibits fat oxidation and rancidity, delays the increase of TVB-N values, and extends the product's shelf life. It is characterized by low cost and high effectiveness. Attached Figure Description

[0011] Figure 1 For the initial screening of nitrite-reducing lactic acid bacteria;

[0012] Figure 2 For secondary screening of nitrite-reducing lactic acid bacteria;

[0013] Figure 3 This is a test to assess the sodium chloride tolerance of lactic acid bacteria.

[0014] Figure 4 This is a test to assess the ethanol tolerance of lactic acid bacteria.

[0015] Figure 5 The degradation curves of nitrite in Lactobacillus fermentum zwt-1 cultured alone and co-cultured with Hansenula d'Bary yeast 2.149 were obtained.

[0016] Figure 6 The effects of Hansenula haematobium 2.149 on the physiological effects of Lactobacillus fermentum zwt-1 were investigated, where A represents the effect on growth kinetics; B represents the effect on pH; C represents the effect on titratable acidity; D represents the effect on lactic acid content; E represents the effect on nitrite reductase activity; and F represents the effect on biofilm formation ability.

[0017] Figure 7Scanning electron microscopy (SEM) images of *Lactobacillus fermentum* zwt-1 cultured alone and co-cultured with *Hansenula d'Barry* 2.149 at different magnifications. A: SEM image of *Lactobacillus fermentum* zwt-1 in KB medium (a1, 3000×; a2, 5000×); B: SEM image of *Lactobacillus fermentum* zwt-1 and *Hansenula d'Barry* 2.149 in KB medium (b1, 3000×; b2, 5000×); C: SEM image of *Lactobacillus fermentum* zwt-1 in GL medium (a1, 3000×; a2, 5000×); D: SEM image of *Lactobacillus fermentum* zwt-1 and *Hansenula d'Barry* 2.149 in GL medium (b1, 3000×; b2, 5000×).

[0018] Figure 8 Optimization of conditions for ultrasonic treatment to degrade nitrite in air-dried duck meat;

[0019] Figure 9 The study investigated the effects of ultrasonic treatment combined with bacterial fermentation on the quality of air-dried duck meat. In this study, A represents the effect on the residual nitrite content of air-dried duck meat, B represents the effect on the nitrite degradation rate, C represents the effect on the pH value of air-dried duck meat, D represents the effect on the thiobarbituric acid value of air-dried duck meat, and E represents the effect on the volatile basic nitrogen value of air-dried duck meat.

[0020] Figure 10 The effects of ultrasonic treatment (US), inoculum fermentation (LD), and ultrasonic treatment combined with inoculum fermentation (US-LD) on the color of duck meat after air drying for 0, 1, 3, 5, and 7 days were investigated. Detailed Implementation

[0021] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments.

[0022] I. Experimental Measurement Methods

[0023] 1. Determination of nitrite degradation rate

[0024] (1) Nitrite degradation rate of bacterial culture: The nitrite content was determined using the Nanjing Jiancheng Nitrite Assay Kit and the nitrite degradation rate was calculated. Nitrite degradation rate / % = [(C0-C1) / C0]*100%, where C0 is the nitrite concentration of the uninoculated GL medium in μmol / L and C1 is the nitrite concentration of the inoculated GL medium in μmol / L.

[0025] (2) Nitrite residue and degradation rate in duck meat

[0026] The residual nitrite in duck meat was determined after air-drying for 7 days. First, 2g of duck meat (excluding tendons and fat) was accurately weighed and added to 20mL of distilled water. The mixture was then homogenized twice at 10000rpm / min for 0.5min each time. Subsequently, it was centrifuged at 8000rpm / min for 10min. The supernatant was then used to determine the nitrite content using the Nanjing Jiancheng Nitrite Rapid Detection Kit.

[0027] 2. pH measurement

[0028] Add 5g of air-dried duck meat to 45mL of deionized water and homogenize twice at 10000r / min for 0.5min each time using a high-speed homogenizer. After standing for 20min, measure the pH value of each duck meat sample using a pH meter.

[0029] 3. Determination of TBARS value of thiobarbituric acid

[0030] Weigh 5g of dried duck meat sample and add 25mL of 10% (v / v) trichloroacetic acid (TCA) solution. Homogenize twice at 10000 rpm for 0.5 min each time using a high-speed homogenizer, then filter through filter paper. Take 2.5mL of the supernatant and mix it with 2.5mL of 0.02mol / L thiobarbituric acid solution. Incubate in a boiling water bath for 40 min, then allow it to cool to room temperature, centrifuge at 5000 rpm for 5 min, take the supernatant, add an equal volume of chloroform, mix well, and allow to separate into layers. Measure the absorbance of the supernatant at 532nm and 600nm, respectively.

[0031] 4. Determination of volatile basic nitrogen (TVB-N) value

[0032] Take 6g of air-dried duck meat and add it to 30mL of sterile water. Homogenize twice using a high-speed homogenizer at 10000r / min, 0.5min each time, then filter with filter paper. Take 10mL of the filtrate, add 5mL of magnesium oxide suspension (10g / L), place it in a digestion tube, and distill using a Kjeldahl nitrogen analyzer for 5min. Add 2-3 drops of mixed indicator to the distillate and titrate with 0.01M hydrochloric acid. Stop the titration when the color changes from light yellow-green to light pink. Mixed indicator: Mix 2 parts methyl red-ethanol solution (1g / L) + 1 part methylene blue-ethanol solution (1g / L), and prepare fresh before use.

[0033] 5. Measurement of color difference

[0034] Color difference was measured using a CR-440 colorimeter to determine the color of duck meat and braising liquid, and expressed by the L* (luminance value), a* (redness value), and b* (yellowness value) parameters in the CIE Labsystem. Before each measurement, the duck meat was cut into uniform thin slices (4cm × 4cm × 2cm) with a thickness of 6 cm. Each meat sample was measured 6 times in parallel. Before testing the meat samples, the standardized white plate of the colorimeter (L* = 94.93, a* = -0.24, and b* = 2.99) was measured with a D65 light source and observed at a 10° angle. II. Specific Implementation Methods

[0036] Example 1

[0037] Preliminary screening of nitrite-degrading lactic acid bacteria according to this invention

[0038] Twelve strains of lactic acid bacteria were isolated from the microbial culture bank of the College of Food Science and Engineering, Ningbo University. These strains are: *Lactobacillus reuteri max*, *Lactobacillus fermentum*, *Lactobacillus fermentum 55*, *Lactobacillus brevis X11*, *Lactobacillus fermentum JF6*, *Lactobacillus fermentum JF9*, *Lactobacillus acidophilus 6074*, *Lactobacillus casei 1.5956*, *Lactobacillus fermentum zwt-1*, and *Lactobacillus brevis* 1.5954. The strains used were *Lactobacillus plantarum* 1.5954, *Lactobacillus plantarum* 1.5953, and *Lactococcus lactis* 5805. For use, each strain was removed from a -80°C freezer, streaked onto MRS agar, and incubated at 37°C for 36 hours. Single colonies from the MRS agar were then inoculated twice into MRS broth to obtain activated strains. The activated lactic acid bacteria were inoculated into modified MRS liquid medium (GL) (the modified MRS liquid medium was prepared as follows: NaNO2 was dissolved in sterile deionized water and filtered through a 0.22 μm filter to obtain a standard stock solution with a concentration of approximately 2500 mg / L. This stock solution was then mixed with sterilized MRS broth at a volume ratio of 1:9 to prepare a modified MRS broth containing 250 mg / L NaNO2). Uninoculated GL medium was used as a blank control. The mixture was incubated at 37°C for 24 h. At the end of the incubation, the nitrite content was determined using a nitrite assay kit, and the nitrite degradation rate was calculated.

[0039] like Figure 1 As shown, four lactic acid bacteria strains exhibited nitrite degradation rates exceeding 70% in GL medium. Among them, Lactococcus lactis 5805 and Lactobacillus plantarum 1.5953 had degradation rates as high as 96.51% and 95.71%, respectively, while Lactobacillus fermentum zwt-1 and Lactobacillus brevis 1.5954 achieved degradation rates of 76.25% and 86.30%, respectively.

[0040] Lactococcus lactis 5805 was purchased from the China General Microbiological Culture Collection Center, with accession number CGMCC No. 5805.

[0041] Lactobacillus plantarum 1.5953 is strain PDD-1 of Lactobacillus plantarum subsp. plantarum, which was deposited at the China General Microbiological Culture Collection Center on June 19, 2018, with the accession number CGMCC No.15953.

[0042] Lactobacillus fermentum zwt-1 is strain PDD-4 of Lactobacillus fermentum, which was deposited at the China General Microbiological Culture Collection Center on March 25, 2019, with accession number CGMCC No. 17434.

[0043] Lactobacillus brevis CGMCC 1.5954 is strain PDD-2 of Lactobacillus brevis, which was deposited on June 19, 2018 at the China General Microbiological Culture Collection Center, with accession number CGMCCNo.15954, located at No.3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences.

[0044] Example 2

[0045] Secondary screening of nitrite-degrading lactic acid bacteria

[0046] Four lactic acid bacteria strains with outstanding nitrite degradation capabilities, selected from Example 1, were further investigated for their nitrite degradation when co-cultured with yeast. Lactic acid bacteria (1%, v / v) and yeast (1%, v / v) were simultaneously inoculated into GL medium, with uninoculated GL medium serving as a blank control. The cultures were incubated at 37°C for 24 hours. Figure 2 As shown, co-culturing with *Hansenula d'Barry* further improved the nitrite degradation rate of all four lactic acid bacteria strains. *Lactobacillus fermentum* zwt-1 showed the highest degradation rate increase of 23.33%, and its nitrite degradation rate was also the highest at 99.58%. Meanwhile, the nitrite degradation rates of *Lactobacillus brevis* 1.5954, *Lactobacillus plantarum* 1.5953, and *Lactococcus lactis* 5805 also increased by 12.43%, 2.78%, and 2.26%, respectively. Further tolerance tests to sodium chloride and ethanol were conducted on these four lactic acid bacteria strains. Figure 3 and Figure 4As shown, with increasing sodium chloride and ethanol concentrations, the growth of *Lactobacillus fermentum* zwt-1, *Lactobacillus brevis* 1.5954, *Lactobacillus plantarum* 1.5953, and *Lactococcus lactis* 5805 was significantly inhibited. When the sodium chloride concentration was 12% and the ethanol concentration was 16%, almost no growth was observed in any of the strains. However, *Lactobacillus fermentum* zwt-1 showed higher tolerance at sodium chloride concentrations of 8% and 12% and ethanol concentrations of 12%. Therefore, *Lactobacillus fermentum* zwt-1 is more suitable for use in the later stages of fermenting air-dried duck meat.

[0047] Further research was conducted on the effect of Hansenula d'Barry yeast 2.149 on the degradation of nitrite by Lactobacillus fermentum zwt-1, such as... Figure 5 As shown, the nitrite degradation rate of *Lactobacillus fermentum* zwt-1 cultured alone increased slowly from 4 hours, reaching a maximum degradation rate of 76.25% at 16 hours, and then remained stable. When co-cultured with *Hansenula baileyi*, the nitrite degradation rate increased rapidly from 4 hours, reaching a maximum degradation rate of 99.58% at 8 hours, and then remained stable. Therefore, co-culturing *Lactobacillus fermentum* zwt-1 with *Hansenula baileyi* 2.149 not only improves the nitrite degradation rate but also demonstrates the ability to rapidly degrade nitrite. *Hansenula baileyi* 2.149 was purchased from the Guangdong Provincial Microbial Culture Collection Center, with accession number GDMCC No. 2149.

[0048] Example 3

[0049] Effects of Hansenula d'Bary yeast 2.149 on the growth and physiological effects of Lactobacillus fermentum zwt-1: Lactobacillus fermentum zwt-1 (2%, v / v) was inoculated into GL medium (modified MRS liquid medium with added sodium nitrite, see Example 1) and incubated at 37°C for 24 h, designated as the GLLS group.

[0050] Lactobacillus fermentum zwt-1 (2%, v / v) was inoculated into KB medium (MRS liquid medium, without sodium nitrite) and incubated at 37°C for 24 h, and was recorded as KBLS group.

[0051] Lactobacillus fermentum zwt-1 (2%, v / v) and Hansenula d'Bary yeast 2.149 (1%, v / v) were co-inoculated into GL medium and incubated at 37°C for 24 h, and were designated as GLLSHS group.

[0052] Lactobacillus fermentum zwt-1 (2%, v / v) and Hansenula d'Bary yeast 2.149 (1%, v / v) were co-inoculated into KB medium and incubated at 37°C for 24 h, and were designated as the KBLSHS group.

[0053] Fermentation broth was collected every 4 hours from each group starting at 0 h for analysis. The viable count of *Lactobacillus fermentum* zwt-1 during co-culture was determined by plate counting on MRS agar containing 10 mg / L nystatin to inhibit the growth of *Hansenula haemolyticus* 2.149.

[0054] The pH value of the fermentation broth was measured using a pH meter.

[0055] The total titratable acidity (TTA) of the fermentation broth is expressed as the amount of 0.1 mol / L NaOH consumed.

[0056] The lactic acid content of the fermentation broth was determined using the p-hydroxybiphenyl method.

[0057] Nitrite reductase (NIR) activity was measured using a kit manufactured by Suzhou Kemin Biotechnology Co., Ltd. (Suzhou, Jiangsu, China).

[0058] like Figure 6 A shows the growth dynamics of *Lactobacillus fermentum* zwt-1 cultured alone and in co-culture in KB and GL media, respectively. Figure 6 As shown in Figure A, the changes in the number of *Lactobacillus fermentum* zwt-1 cells in KB and GL media were measured after 24 hours of single and co-culture. *Hansenula polymorpha* 2.149 significantly affected the growth of *Lactobacillus fermentum* zwt-1. In the co-culture systems of KB and GL media, the number of *Lactobacillus fermentum* zwt-1 cells differed significantly with increasing fermentation time compared to single culture. Moreover, in KB media, the number of *Lactobacillus fermentum* zwt-1 cells was dominant in both single and co-culture systems, possibly because the presence of nitrite in GL media inhibited the activity of *Lactobacillus fermentum* zwt-1. With increasing fermentation time, the number of *Lactobacillus fermentum* zwt-1 cells in the co-culture system was significantly higher than that in the single culture system. Specifically, the number of *Lactobacillus fermentum* zwt-1 cells cultured alone in KB and GL media were 9.40 and 8.71 log CFU / mL, respectively, while the number of *Lactobacillus fermentum* zwt-1 cells in the co-culture system at 24 hours were 9.70 and 9.34 log CFU / mL, respectively. The increased number of *Lactobacillus fermentum* zwt-1 cells may be due to their non-competitive use of nitrogen sources and the secretion of compounds by yeast during metabolism that stimulate lactic acid bacteria growth. Under conditions where organic and inorganic nitrogen sources coexist, yeast preferentially utilizes the latter, while lactic acid bacteria preferentially utilize free amino acids or small peptides. During yeast growth or accelerated autolysis, yeast secretes essential amino acids that may be required for the growth of *Lactobacillus fermentum* (LAB).

[0059] Figure 6 B and Figure 6C shows the changes in pH and total titratable acidity (TTA) of *Lactobacillus fermentum* zwt-1 and *Hansophila baryensis* 2.149 in single and co-culture systems in KB and GL media, respectively. With prolonged fermentation time, the pH of *Lactobacillus fermentum* zwt-1 in both single and co-culture systems in KB and GL media continuously decreased, while the TTA value continuously increased. The results indicate that the decrease in pH and the increase in TTA are consistent. Furthermore, compared with the pH of the fermentation broth in KB medium, the pH of both single and co-culture fermentation broths in GL medium was significantly higher. Correspondingly, the TTA value of the fermentation broth in GL medium also decreased significantly. This phenomenon may be related to the inhibition of lactic acid bacteria growth by nitrite. Notably, in GL medium, starting from 4 hours, the pH of the co-culture system was significantly lower than that of the single culture system, and correspondingly, the TTA value of the co-culture system was significantly higher than that of the single culture system. This change may be related to the increased number of *Lactobacillus fermentum* zwt-1 cells in the co-culture system. The interaction between *Lactobacillus fermentum* zwt-1 and *Hansophila baryana* 2.149 may promote the growth of lactic acid bacteria, thereby further promoting the production of organic acids. Furthermore, the study found that the TTA value of *Lactobacillus fermentum* cultured alone in KB medium was significantly higher than that in the co-culture system in GL medium within 4–20 h. After 20 h, the TTA value began to decrease, and at 24 h, there was no significant difference in TTA values ​​between the two. This may be because after 20 h, the low pH in KB medium led to the decline of lactic acid bacteria and a decrease in cell number. In the GL medium co-culture system, the yeast captured the information of acid production by the lactic acid bacteria. To adapt to sub-lethal acid concentrations and grow, they formed a mixed-species biofilm with the lactic acid bacteria, which increased the lactic acid bacteria's tolerance to acidic environments and nitrite.

[0060] Figure 6 Figure D shows the changes in lactic acid content during fermentation in KB and GL media, both individually and in co-culture. The results showed that the lactic acid content in KB medium was significantly higher than that in GL medium from 4 h to 20 h, indicating that nitrite inhibited lactic acid production by *Lactobacillus fermentum* zwt-1. Compared to the lactic acid content of *Lactobacillus fermentum* zwt-1 cultured alone in KB medium, its lactic acid production was greatly inhibited in GL medium, while the lactic acid content in the co-culture system with *Hansenula polymorpha* 2.149 increased slowly, indicating that *Hansenula polymorpha* 2.149 can promote lactic acid production by *Lactobacillus fermentum* zwt-1.

[0061] Nitrite reductase is a class of enzymes that catalyze the reduction of nitrite, degrading it into NO or NH3. It is a key enzyme in the nitrogen cycle in nature. The nitrite reductase activity after 24 hours of fermentation is as follows: Figure 6As shown in Figure E, the GLLS group exhibited the highest nitrite reductase activity, reaching 4.79 U / mg at 24 h, significantly higher than the other three groups. The enzyme activities of KBLS, KBLSHS, and GLLSHS were 1.67, 1.54, and 1.29 U / mg, respectively, with no significant difference (P>0.05). Nitrite reductase activity is affected by pH; pH < 5.0 leads to a sharp decrease in enzyme activity. This may be one reason for the lower enzyme activity in these three groups.

[0062] Example 4

[0063] Effects of Hansenula d'Barry 2.149 on the biofilm formation ability of Lactobacillus fermentum zwt-1. Biofilm is a highly organized microbial community structure. Biofilm formation improves the survival rate of most bacteria and also helps bacteria resist high temperature, inappropriate pH, osmotic pressure, metal ions, antibiotics and other microorganisms. Figure 6 F shows the changes in biofilm content when *Lactobacillus fermentata* zwt-1 and *Baryia hansenulatus* 2149 were cultured individually and co-cultured in KB and GL media, respectively. Figure 6 As shown in Figure F, different culture conditions (KB and GL media) had no significant effect on biofilm formation of *Lactobacillus fermentum* zwt-1 and *Hansøe Baryella* 2.149, or when both were co-cultured (P>0.05). After 24 hours of culture, the biofilm content of *Hansøe Baryella* 2.149 was significantly higher than that of *Lactobacillus fermentum* zwt-1 (P<0.05). However, the biofilm content of both *Lactobacillus fermentum* zwt-1 and *Hansøe Baryella* 2.149 cultured alone was significantly lower than that of co-cultured (P<0.05). Microorganisms exhibit strong tolerance to environmental stress and antibiotics and can survive under adverse conditions in the form of biofilms. During food fermentation, they can also exist in the form of biofilms, which has a significant impact on the quality and flavor of fermented foods. Therefore, mixed biofilms may be a possible lifestyle for *Lactobacillus fermentum* and *Hansøe Baryella* in traditional starter cultures. Hansenula d'Bary yeast 2.149 is the main biofilm producer. The biofilm content in the co-culture system of Hansenula d'Bary yeast 2.149 is higher than that in the monoculture system, which may be due to the formation of mixed species biofilm in the co-culture system. This may be related to the increase in the number of Lactobacillus fermentum zwt-1 cells in the co-culture system.

[0064] Figure 7 The morphology of *Lactobacillus fermentum* zwt-1 and *Hansenula polymorpha* 2.149 under different magnifications during single and co-culture in KB and GL media, respectively. *Lactobacillus fermentum* zwt-1 showed no significant morphological changes during single and co-culture in KB and GL media; the cells were elongated rod-shaped, plump, with a smooth surface, approximately 0.9-1.2 μm long and 0.3-0.4 μm wide. Figure 7A, C). When co-cultured in GL medium, the surface of *Hansenula haematocephala* 2.149 showed slight wrinkling (…). Figure 7 D). It is worth noting that in the co-culture electron micrographs ( Figure 7 In B and D), we observed that Lactobacillus fermentum and Hansson's Barry yeast 2.149 existed in contact, with Lactobacillus fermentum zwt-1 surrounding Hansson's Barry yeast 2.149 and interacting with each other. This is consistent with our previous conclusions, as contact culture between Lactobacillus fermentum zwt-1 and Hansson's Barry yeast 2.149 resulted in the highest biofilm content in the co-culture system.

[0065] Example 5

[0066] Preparation and optimization of ultrasonic conditions for air-dried duck meat

[0067] 1. Raw materials: Thaw frozen duck embryos under running water for 0.5 hours, trim and clean them, and drain the water;

[0068] 2. Pickling solution: The pickling solution is a mixture of 8 wt% sodium chloride and 0.01 wt% sodium nitrite;

[0069] 3. Marinating: Marinate with 1100-1200mL of marinade per kilogram of duck meat. The duck embryo should be completely submerged in the liquid during marinating. The marinating time is 24 hours and the marinating temperature is 4℃.

[0070] 4. Use an ultrasonic cleaner at 0W, 150W, 200W, 250W, 300W, 350W, and 400W respectively to ultrasonicate the marinated duck meat for 1 minute, 2 minutes, 3 minutes, and 4 minutes respectively, and then air dry it.

[0071] 5. Air-drying fermentation: The fermentation agent (activated Lactobacillus fermentum zwt-1 and Hansenula d'Barry yeast 2.149 washed three times with sterile physiological saline, adjusting the cell concentration OD600 to 1.0, inoculation ratio 1:1, v / v) is evenly sprayed onto the surface of the duck embryos, with an inoculation amount of 10. 7 CFU / g, constant temperature and humidity chamber program temperature 16℃, relative humidity 68%, air drying for 7 days.

[0072] like Figure 8 As shown, the residual nitrite content of all samples after ultrasonic treatment was significantly lower than that of samples without ultrasonic treatment. Among them, the sample ultrasonicated at 250W for 4 minutes had the lowest nitrite content, and its residual nitrite content was 2.89 mg / kg after air drying for 7 days.

[0073] III. Analysis of Experimental Results

[0074] The application of *Lactobacillus fermentum* zwt-1 selected in the above specific embodiment one, along with purchased *Hansophila barleyi* 2.149 and ultrasonic treatment in air-dried duck meat, was investigated. This invention used sonication alone (250W, 4min, US), inoculation fermentation alone (*Lactobacillus fermentum* zwt-1 + *Hansophila barleyi* 2.149 1:1, LD), and their combinations (US-LD), with naturally fermented duck embryos as a blank control (CK). The application in air-dried duck meat was explored, and samples were taken at 0d, 1d, 3d, 5d, and 7d after air-drying to determine nitrite content, pH, TBARS, TVB-N, and color difference. The sample preparation method is as follows, referring to Example 5 in the specific embodiments: CK group samples were left untreated after marinating; US group samples were ultrasonically cleaned at 250W for 4 minutes after marinating; LD group samples were dried after marinating, and the fermentation agent (Lactobacillus fermentum zwt-1 + Hansenula d'Barry yeast 2.1491:1) was evenly sprayed onto the surface of the duck embryos at an inoculation amount of 10... 7 After marinating, the CFU / g and US-LD group samples were first ultrasonically treated and dried. Then, the fermentation agent was evenly sprayed onto the surface of the duck embryos at an inoculation amount of 10. 7 CFU / g.

[0075] 1. Nitrite residue and degradation rate in air-dried duck meat.

[0076] like Figure 9 As shown in Figures A and B, during the air-drying process, the residual nitrite content in the air-dried duck meat of group CK showed a trend of first decreasing and then increasing. This may be because some microorganisms carried by the duck embryo itself may have the ability to reduce nitrite, thereby promoting nitrite degradation. However, with the extension of air-drying time, the activity of other microorganisms may, to some extent, cause nitrite to reform, leading to a renewed increase in its content. In contrast, the residual nitrite content in air-dried duck meat treated with ultrasound, inoculated fermentation, and a combination of both showed a gradual decreasing trend during the air-drying process. This may be because ultrasound treatment utilizes the cavitation effect of ultrasound to generate tiny bubbles in the meat product. These bubbles then collapse instantaneously, generating intense local high temperature and high pressure that destroys cell structure, which is conducive to promoting the decomposition reaction of nitrite. This treatment method is more efficient at removing nitrite in the early stages, which may be one of the reasons why the residual nitrite content of ultrasound treatment was lower than that of inoculated fermentation treatment on the first day of air-drying. However, in the later stages of air-drying, due to the continuous acid production by lactic acid bacteria during fermentation, the pH value continuously decreases, and the nitrite in the meat product reacts with H+. +A reversible disproportionation reaction occurs, generating nitrates, leading to a significant decrease in nitrite content. After 7 days of air drying, the residual nitrite in the ultrasonic treatment combined with bacterial fermentation group was 0.91 mg / kg, with a degradation rate of 84.55%. This is significantly lower than the 5.92 mg / kg in the control group, 2.57 mg / kg in the ultrasonic treatment alone group, and 1.90 mg / kg in the bacterial fermentation alone group. This may be because ultrasonic treatment of duck meat causes the cell membranes of the microorganisms it carries to rupture, making it easier for the strains to absorb nutrients and metabolites from the surrounding environment after inoculation, thus enhancing the metabolic activity of the strains. In addition, ultrasonic treatment can promote the transfer and diffusion of substances in meat products, making it easier for the strains to come into contact with nitrites in duck meat, accelerating their degradation and metabolic processes. The results indicate that using ultrasonic treatment combined with bacterial fermentation can more effectively degrade the nitrite content in fermented foods, ensuring the safety of fermented air-dried duck for consumption.

[0077] 2. The effect of pH value on air-dried duck meat

[0078] pH is an important indicator of meat product quality. Changes in pH affect the interactions between proteins, thereby influencing protein structure and other functional properties. Figure 9 As shown in Figure C, during the air-drying process, the pH values ​​of the CK and US groups gradually increased, while the pH values ​​of the LD and US-LD groups gradually decreased. The pH of the air-dried duck meat treated with ultrasound showed an increasing trend compared to the CK and US-LD groups, indicating that ultrasound treatment improved the pH value of the dried duck meat. This may be because ultrasound treatment causes cell membrane damage and the release of intracellular ions, or induces protein structural modifications, leading to the exposure of basic groups in the proteins, thus increasing the pH. The air-dried duck meat treated with LD had the lowest pH value, which may be attributed to the organic acids produced by LAB. Low pH values ​​are beneficial for promoting the degradation of nitrite in meat products, inhibiting the growth of spoilage bacteria, and extending the shelf life of meat products.

[0079] 3. The effect of TBARS on the thiobarbituric acid value of air-dried duck meat

[0080] The TBARS value in fermented meat products reflects the degree of fat oxidation and is an important indicator of their quality. This invention investigates the effects of different treatments on fat oxidation in air-dried duck meat by measuring the TBARS values ​​of different fermentation groups. The results are as follows: Figure 9As shown in Figure D, the TBARS values ​​of all groups of air-dried duck meat showed a gradual upward trend during the air-drying process. The US group exhibited the largest change, rising from 0.05 mg MDA / 100g at the beginning of air-drying to 0.32 mg MDA / 100g at the end. This may be because ultrasonic treatment alters the protein structure, especially pigment proteins such as hemoglobin and myoglobin, leading to the release of iron ions and other transition metal ions, which can increase the level of lipid oxidation. Simultaneously, the cavitation effect of ultrasound can break down water molecules, generating highly oxidizing hydroxyl radicals, which may also be a major cause of lipid oxidation in air-dried duck meat. Compared with the CK group, the TBARS values ​​of the US and US-LD groups showed smaller changes and were significantly lower than those of the CK group in the later stages of air-drying (P<0.05). This indicates that co-fermentation with *Lactobacillus fermentum* zwt-1 and *Baryia hansenii* 2.149 has an inhibitory effect on lipid oxidation in air-dried duck meat. This may be because Lactobacillus fermentum zwt-1 and Hansenula d'Barry yeast 2.149 can inhibit the growth of hydrogen peroxide-producing strains in dried duck meat or have strong catalase, which can decompose the hydrogen peroxide produced by dried duck meat.

[0081] 4. Effect of TVB-N on the volatile basic nitrogen value of air-dried duck meat

[0082] Volatile basic nitrogen (VBNi) is the alkaline nitrogenous substance produced by the putrefaction of proteins in animal-based foods under the action of enzymes and bacteria, resulting in ammonia and amines. This value is an important indicator for judging the freshness of meat products; a higher VBNi content in dried duck meat indicates a higher degree of protein putrefaction. For example... Figure 9As shown in Figure E, the TVB-N content of each group of samples gradually increased with the extension of air-drying time. Throughout the air-drying process, the TVB-N values ​​of US, LD, and US-LD were significantly lower than those of the CK group (P<0.05). At the end of air-drying, the TVB-N values ​​of US, LD, and US-LD were 29.17 mg / 100g, 26.37 mg / 100g, and 21.47 mg / 100g, respectively, significantly lower than the 35.23 mg / 100g of the CK group (P<0.05). This indicates that ultrasonic treatment and co-fermentation with *Lactobacillus fermentum* zwt-1 and *Hansøe Baryella* 2.149 both inhibited protein degradation in air-dried duck meat. This may be because ultrasonic treatment killed some microorganisms in the duck meat, inhibiting their growth and reproduction, thereby delaying the increase in TVB-N value and slowing down product spoilage. Inoculating duck meat with *Lactobacillus fermentum* zwt-1 and *Hansophila hansun d'Barry* 2.149 rapidly established them as dominant strains, inhibiting the growth of spoilage bacteria and producing large amounts of lactic acid. This lactic acid reacted with ammonia or amines produced by bacteria in the duck meat, reducing the TVB-N value. During the air-drying process, the US-LD group consistently exhibited the lowest TVB-N value. This is likely because ultrasonic treatment improved the structure and properties of the meat products, making it easier for the strains to penetrate and exert their effects, increasing their metabolic activity and efficiency, and more effectively inhibiting the formation of nitrogen compounds in the meat products, thus synergistically suppressing the increase in TVB-N value.

[0083] 5. Effects of ultrasonic treatment combined with bacterial fermentation on the color of air-dried duck meat

[0084] Color is a direct sensory factor for consumers regarding dried duck meat, influencing their acceptance of it. After removing the surface muscle tissue from the dried duck meat, the color of the internal cross-section was measured. A spectrophotometer was used to measure the L* (brightness), a* (redness), and b* (yellowness) of the dried duck meat. Before the experiment, the colorimeter was standardized and calibrated using a white standard plate (L* = 94.07, a* = 0.17, b* = 0.24). Because the color of duck meat varies slightly from different parts, three parallel measurements were taken from different locations for each piece of duck meat, and the average value was calculated. The results are as follows: Figure 10 As shown in Table 1.

[0085] Table 1. Effects of different treatment methods on the color of air-dried duck meat

[0086]

[0087]

[0088] Table 1 shows that the brightness value (L*) of the four groups of air-dried duck meat initially decreased and then increased with drying time, but the CK group consistently showed the highest L*. The CK group's L* reached its minimum value of 36.16 on day 3, significantly higher than the US, LD, and US-LD groups (33.48, 31.86, and 30.94, respectively). This indicates that ultrasonic treatment and co-fermentation with *Lactobacillus fermentum* zwt-1 and *Hansophila bleachys* 2.149, or their combination, are not conducive to improving the L* of air-dried duck meat. The redness value (a*) of air-dried duck meat has a significant impact on its color. Table 1 shows that the a* of each group of air-dried duck meat initially increased and then slowly decreased. At 3 days of air drying, the a* values ​​of each group reached their maximum, at 14.80, 15.57, 17.33, and 18.95, respectively. The a* values ​​of the CK and US groups showed no significant difference (P>0.05), but were significantly lower than the other two groups (P<0.05). Subsequently, with prolonged air drying, the a* values ​​gradually decreased. At the end of air drying, the a* values ​​of the LD and US-LD groups were 13.26 and 15.02, respectively, both significantly higher than the US group's 11.16 and the CK group's 9.80 (P<0.05). This indicates that co-fermentation of *Lactobacillus fermentum* zwt-1 and *Hansøe du Barry yeast* 2.149 contributes to the increase of a* in air-dried duck meat. This may be because the strain decomposes nitrite in the meat into NO, and NO reacts with myoglobin (Mb) in the meat to generate nitrosomyoglobin (MbNO), promoting the color development of the duck meat. As shown in Table 1, during the air-drying process, the yellowness value (b*) of the air-dried duck meat in each group showed a trend of first decreasing and then increasing. The b* of the air-dried duck meat after ultrasonic treatment was significantly higher than that of the other three groups. After air-drying, the b* of the US group was 15.53, significantly higher than that of the CK group, LD group, and US-LD group (12.55, 10.62, and 11.45, respectively, P<0.05). This may be because ultrasonic treatment promotes the formation of free radicals. Hydroxyl radicals are reactive oxygen species that can undergo peroxidation reactions with polyunsaturated fatty acids, accelerating the oxidation process of fats. This is consistent with our previous finding that ultrasonic treatment can increase the TBARS value.

[0089] The foregoing description is not intended to limit the invention, nor is the invention limited to the examples given. Any changes, modifications, additions, or substitutions made by those skilled in the art within the scope of the invention should also be considered within the protection scope of the invention.

Claims

1. A method for producing fermented duck with low nitrite residue, characterized in that... Includes the following steps: Marinate the washed and drained duck meat in 1100–1200 mL of marinade per kilogram of duck meat at a temperature of 0–4°C for 20–28 hours. After marinating, ultrasonically clean the duck meat at 150–400 W for 1–4 minutes, then air dry. Evenly spray the surface of the duck embryo with a starter culture consisting of *Lactobacillus fermentum* and *Hansenula polymorpha* 2.149 (accession number GDMCC No. 2149), at an inoculation rate of 10. 6 ~10 8 CFU / g, placed in a constant temperature and humidity chamber at 14-18℃ and 65-70% relative humidity, and air-dried for 6-8 days, yields fermented duck with low nitrite residue. The fermenting lactobacillus mentioned is *Lactobacillus fermentum* (CFU / g). Lactobacillus fermentum PDD-4 strain was deposited at the China General Microbiological Culture Collection Center (CGMCC) on March 25, 2019, with accession number CGMCC No. 17434.

2. The method for producing fermented duck with low nitrite residue according to claim 1, characterized in that: The pickling solution is a mixture containing 8 wt% sodium chloride and 0.01 wt% sodium nitrite.

3. The method for producing fermented duck with low nitrite residue according to claim 1, characterized in that... The preparation method of the starter culture is as follows: The activated Lactobacillus fermentum and Saccharomyces hanseniifolius were washed three times with sterile physiological saline, and the cell concentration (OD) was adjusted accordingly. 600 =1.0, then mixed by volume at a ratio of 1:

1.

4. The method for producing fermented duck with low nitrite residue according to claim 1, characterized in that... The ultrasonic processing power is 250W and the ultrasonic time is 4 minutes.

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