Yeast utilizing non-protein nitrogen and application thereof
By developing the Candida yeast strain that produces Candida FJM60 and using non-protein nitrogen sources for solid fermentation, the problem of low microbial utilization efficiency in the existing technology has been solved, and efficient protein production and improved feed nutritional value has been achieved.
Patent Information
- Application Number
- CN202411673117.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-21
- Publication Date
- 2025-03-11
AI Technical Summary
In the prior art, there are fewer types of microorganisms that utilize non-protein nitrogen and are not very efficient in utilization, resulting in low protein production efficiency and resource shortage.
A new yeast strain, Candida primrose FJM60 (CGMCC No. 31888), was developed, which can grow efficiently in a variety of non-protein nitrogen sources and improve the nutritional value of the feed through solid-state fermentation technology.
FJM60 yeast shows high growth rate, viable bacteria number and protein synthesis efficiency when utilizing non-protein nitrogen sources, which significantly improves the crude protein and real protein content of the feed, improves the physical structure of the feed and animal digestibility, and enhances the health and environmental protection of the animal.
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Abstract
Description
Technical Field
[0001] The present invention relates to the field of microbial technology, in particular to a yeast utilizing non-protein nitrogen and application thereof. Background Art
[0002] As the global demand for protein resources continues to increase, non-protein nitrogen (NPN) as a potential alternative protein resource is gradually attracting attention from the scientific community. Non-protein nitrogen mainly includes compounds such as urea, ammonia, ammonium salts and amides. These substances are widely present in nature, but are often ignored in traditional protein production.
[0003] At present, there are relatively few microorganisms that can utilize non-protein nitrogen, and the utilization efficiency is generally low. Therefore, developing a microbial resource that can efficiently utilize non-protein nitrogen is of great significance for improving protein production efficiency and alleviating the shortage of protein resources. Summary of the invention
[0004] In view of this, the present invention proposes a yeast that utilizes non-protein nitrogen and an application thereof, aiming to develop a new microbial resource that can efficiently utilize non-protein nitrogen.
[0005] The present invention provides a yeast that utilizes non-protein nitrogen, and the yeast that utilizes non-protein nitrogen is named Candida utilis FJM60;
[0006] The deposit number of the Candida utilis FJM60 is CGMCC No.31888.
[0007] The present invention also proposes an application of the yeast utilizing non-protein nitrogen, wherein the application comprises at least one of the following:
[0008] A1) Application in fermented wheat bran;
[0009] A2) Application in fermentation of distiller's grains;
[0010] A3) Application in the preparation of biological feed additives;
[0011] A4) Application in the preparation of biological feed;
[0012] A5) Application in degradation of organic pollutants;
[0013] A6) Application in improving apparent metabolizable energy of poultry.
[0014] Preferably, the organic pollutant is wheat bran or distiller's grains.
[0015] The present invention also provides a bacterial agent, which comprises the Candida utilis FJM60.
[0016] The present invention also provides a method for preparing bacterial protein feed, comprising:
[0017] The fermentation substrate is fermented by using the Candida utilis FJM60 to obtain the bacterial protein feed.
[0018] Preferably, the fermentation substrate is wine grains and ammonia water.
[0019] Preferably, the fermentation substrates are wheat bran, urea and ammonium sulfate.
[0020] Compared with the prior art, the present invention has the following beneficial effects:
[0021] Successful utilization of non-protein nitrogen sources: The FJM60 disclosed in the present invention can grow efficiently in a variety of non-protein nitrogen source environments and show significant biomass accumulation and protein production capacity, which provides a new idea for solving the problems of high nitrogen source costs and limited resources in the feed industry. Specifically, when FJM60 utilis uses non-protein nitrogen sources such as ammonium sulfate, ammonium chloride, urea and ammonia water, its growth rate, viable count and protein synthesis efficiency are better than other tested strains, showing strong environmental adaptability and resource utilization efficiency.
[0022] Significantly improve the nutritional value of feed: Through the solid-state fermentation process of FJM60 yeast, not only the crude protein content in wheat bran is increased, especially the proportion of true protein is significantly increased, but also the types and contents of amino acids are enriched, which is of great significance for improving the overall nutritional value of feed. At the same time, the reducing sugars and organic acids (such as lactic acid, acetic acid, etc.) produced during the fermentation process help improve the palatability and digestibility of feed, thereby increasing the feed intake and growth performance of animals.
[0023] Optimize feed structure: The crude fiber content of wheat bran after fermentation is reduced, which helps to improve the physical structure of the feed and make it easier for animals to digest and absorb. In addition, the fermentation process may also be accompanied by the degradation of some anti-nutritional factors, further improving the safety and effectiveness of the feed.
[0024] Improve animal health: The beneficial microorganisms and their metabolites produced during the fermentation process can regulate the microecological balance of the animal's intestines, improve animal immunity, and reduce the occurrence of diseases. This is of great significance for improving breeding efficiency and ensuring animal welfare.
[0025] Environmental protection and sustainable development: Converting wheat bran, which might have been waste, into high-value feed through fermentation not only reduces environmental pollution but also achieves the recycling of resources. BRIEF DESCRIPTION OF THE DRAWINGS
[0026] Various other advantages and benefits will become apparent to those of ordinary skill in the art by reading the detailed description of the preferred embodiments below. The accompanying drawings are only for the purpose of illustrating the preferred embodiments and are not to be considered as limiting the present invention. Moreover, the same reference symbols are used throughout the accompanying drawings to represent the same components. In the accompanying drawings:
[0027] Figure 1 The wet weight of centrifuged bacterial sludge after fermentation of 10 screened yeast strains in peptone and ammonia basic medium;
[0028] Figure 2 The protein content of centrifuged bacterial sludge after fermentation of 10 screened yeast strains in peptone and ammonia basic medium;
[0029] Figure 3 The results of the viable bacterial count of 10 screened yeast strains after fermentation in peptone and ammonia basic medium;
[0030] Figure 4 The growth curves of FJM18 and FJM60 in basal and ammonia water medium were measured with samples taken every 2 hours;
[0031] Figure 5 Growth curves of FJM18 and FJM60 in basal and ammonia culture medium sampled every 4 hours. DETAILED DESCRIPTION
[0032] Exemplary embodiments of the present disclosure will be described in more detail below with reference to the accompanying drawings. Although exemplary embodiments of the present disclosure are shown in the accompanying drawings, it should be understood that the present disclosure can be implemented in various forms and should not be limited by the embodiments described herein. On the contrary, these embodiments are provided in order to enable a more thorough understanding of the present disclosure and to fully convey the scope of the present disclosure to those skilled in the art. It should be noted that, in the absence of conflict, the embodiments of the present invention and the features described in the embodiments can be combined with each other. The present invention will be described in detail below with reference to the accompanying drawings and in combination with the embodiments.
[0033] Example 1
[0034] Yeast Isolation and Screening
[0035] 1. Initial screening
[0036] Five samples of naturally fermented feed and three samples of chicken intestinal contents (including jejunum, ileum and cecum) were collected. The samples were screened and sequenced using gradient dilution and selective culture medium technology. In view of the positive correlation between the number of viable bacteria and bacterial protein, we adopted a simple primary screening method. The nitrogen sources such as peptone in the screening culture medium YPD were replaced with ammonium sulfate, ammonium chloride, urea and ammonia water to prepare a solid culture medium. Subsequently, a single colony was selected and inoculated into YPD liquid culture medium and cultured at 30°C and 180rpm for 15 hours. Afterwards, the bacterial solution was diluted to -5 and poured into the plate using the 0.5mL pouring method. After 2-3 days of culture, the colonies were counted.
[0037] A total of 15 yeast strains were isolated in the preliminary screening (see Table 1). Among these 15 strains and 3 laboratory-preserved strains, strain FJM60 Candida utilis was deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration (Beijing, China) on September 5, 2024, with the deposit number CGMCC No.31888. The viable count of strain FJM60 is shown in Table 2.
[0038] Table 1
[0039]
[0040]
[0041] Table 2
[0042] Bacteria YPD Ammonium sulfate Ammonium chloride Urea FJM60 7.711219 7.682618 7.609075 7.095213
[0043] After preliminary screening, we found that 10 strains, including FJM60 (Candida utilis), CJM15 (Saccharomyces cerevisiae), JT2 (Pichia pastoris), FJM18 (Pichia kudriazni), CJM30 (Omerkoda yeast), and FJM20 (Pichia kudriazni), performed well when using ammonia and ammonium sulfate as regulators. During the experiment, we used liquid culture medium to adjust the pH value of the above strains, with an inoculation amount of 1%, and measured their biomass, protein content, and viable count after 72 hours of culture. The results are as follows Figure 1 , Figure 2 and Figure 3 As shown in Figure 2, Candida utilis FJM60 and Pichia kudriavzevii FJM18 have the strongest ability to utilize non-protein nitrogen, with the number of viable cells reaching 10 7 cfu / mL. The changes of sample bacterial protein content, wet weight and viable count were basically consistent.
[0044] The growth curves of FJM18 and FJM60 strains in basal and ammonia culture media were measured by recording data every two hours for 30 hours of continuous observation. To ensure the reliability of the experimental results, two repeated experiments were conducted, with three sets of parallel controls set up in each experiment. Figure 4 and Figure 5 As shown, whether measurements are taken every two hours or every four hours, the data obtained are highly consistent.
[0045] Example 2 Single yeast fermentation experiment
[0046] 1. Single yeast fermentation experiment of distiller's grains
[0047] FJM60 fermented three kinds of Luzhou and Wuliangye (and dried part of the wet lees and crushed them) wet material fermentation, and added yeast solution (ammonia culture medium) at an initial OD value of 0.5. The amount of ammonia added was 100g + 2.75ml ammonia, and the pH was 6.8-7. The data of inoculation amount and viable bacteria count are shown in Table 3. The wet material was sampled once every 48h (20g) and dried. The results of the number of viable yeasts corresponding to the solid-state fermentation time of the single bacteria of Luzhou Laojiao Luzhou-flavor lees are shown in Table 4. The results of the number of viable yeasts corresponding to the solid-state fermentation time of the single bacteria of Wuliangye Luzhou-flavor lees are shown in Table 5.
[0048] Table 3
[0049]
[0050] Table 4
[0051]
[0052] Table 5
[0053]
[0054]
[0055] As shown in Table 3-5, the number of live bacteria of FJM60 strains showed different trends during the fermentation process on Luzhou Laojiao and Wuliangye lees. On Luzhou Laojiao lees, the number of live bacteria of FJM60 strains reached a peak of 2.19×10 at 48 hours. 8 cfu / mL, and then decreased, but remained at 4.9×10 7 cfu / mL. On Wuliangye distiller’s grains, the number of viable bacteria of FJM60 strain was 1.33×10 6 cfu / mL, which was lower than that of Luzhou Laojiao distiller's grains. The number of live bacteria decreased after 72 hours, but recovered to 4.9×10 at 120 hours. 7cfu / mL.
[0056] In the control group of natural fermentation, 9 bacteria appeared on the Luzhou Laojiao lees at 48 hours, while several foreign bacteria appeared on the Wuliangye lees at 72 hours, indicating that the growth of foreign bacteria was inhibited during natural fermentation, while the fermentation process of FJM60 strain was more active.
[0057] During the fermentation process, the emergence of foreign bacteria may be related to the control of the fermentation environment, such as pH, temperature, humidity and other factors, which may affect the competition and growth of microorganisms during the fermentation process. At 96 hours, the FJM60 strain fermented samples on Luzhou Laojiao lees had a bad smell, which may be related to the growth of foreign bacteria, while the samples on Wuliangye lees had a fragrance at the same time point, which may indicate that the fermentation process of FJM60 strain on Wuliangye lees is more ideal.
[0058] In summary, there are differences in the fermentation effects of FJM60 on Luzhou Laojiao and Wuliangye distiller's grains, which may be related to the raw material characteristics of the distiller's grains, fermentation conditions and the adaptability of the strains. In practical applications, appropriate strains should be selected and fermentation processes should be optimized according to specific fermentation goals and conditions.
[0059] The crude protein of FJM60 was tested. The results of Luzhou Laojiao Luzhou-flavor type are shown in Table 6, the results of Wuliangye Luzhou-flavor type are shown in Table 7, and the results of Sichuan Langjiu Luzhou-flavor type are shown in Table 8
[0060] Table 6
[0061]
[0062]
[0063] Table 7
[0064]
[0065] Table 8
[0066] index Raw materials, strains Raw materials are not fermented FJM60 powder, 50% 48h Viable bacteria count (cfu / mL) <![CDATA[2.69×10 8 ]]> 48h Crude protein% 13.745 16.92 96h Viable bacteria count (cfu / mL) <![CDATA[Contaminating bacteria, 6×10 6 > 96h Crude protein% 16.598
[0067] As shown in Tables 6-8, the mannan and crude protein contents of the three different lees of Luzhou Laojiao, Wuliangye and Sichuan Langjiu after fermentation with FJM60 strains changed. On the lees of Luzhou Laojiao, the mannan content was 0.345% at 48 hours, and slightly increased to 0.379% at 96 hours, while the crude protein content increased from 12.392% to 15.602%. The crude protein content of Wuliangye lees increased from 11.927% to 17.441%. The crude protein content on the lees of Sichuan Langjiu increased from 13.745% to 16.598%.
[0068] The increase in crude protein content indicates that the FJM60 strain effectively converted the nutrients in the grains during the fermentation process, increasing the nutritional value of the product.
[0069] Taking into account the changes in crude protein content, it can be concluded that the fermentation effects of FJM60 strains on different distiller's grains have their own characteristics. The crude protein content of Luzhou Laojiao distiller's grains after fermentation has increased most significantly. The crude protein content of the products fermented from Sichuan Langjiu distiller's grains has shown a good improvement effect. Therefore, in practical applications, appropriate distiller's grains raw materials and fermentation strains can be selected according to different needs to achieve the purpose of optimizing the nutritional value of feed.
[0070] In addition, the change in the number of live bacteria during the fermentation process also reflects the adaptability and fermentation efficiency of FJM60 strains on different lees. The peak number of live bacteria of FJM60 strains on Luzhou Laojiao lees reached a peak at 48 hours, while the peak number of live bacteria on Wuliangye lees appeared at 96 hours, which may be related to the characteristics of the lees raw materials. The fermentation effect on Sichuan Langjiu lees was between the two, indicating that FJM60 strains have good adaptability to different lees raw materials.
[0071] In summary, the results of the fermentation experiment of FJM60 strain on different distiller's grains showed that the strain had good fermentation potential and application value. By optimizing the fermentation conditions and process, the fermentation efficiency and product quality can be further improved, providing a scientific basis for the production of bacterial protein feed with high nutritional value.
[0072] 2. Single yeast fermentation experiment of wheat bran
[0073] 3% urea and 3% ammonium sulfate were added as nitrogen sources to the wheat bran treated by the FJM60 fermentation process. Under the condition of an initial OD value of 0.5, an appropriate amount of yeast liquid was added and the fermentation process lasted for 48 hours. Subsequently, the wet material after fermentation was dried. The specific contents of dry matter recovery, crude protein, true protein and other nutrients of the fermentation group with 3% urea and 3% ammonium sulfate are shown in Table 9.
[0074] Table 9
[0075]
[0076] 3. Summary
[0077] Using wheat bran and distiller's grains as substrates, the growth characteristics of FJM60 yeast strain under different conditions and its improvement effect on nutritional components were studied by solid-state fermentation. The following is a further analysis and summary of the experimental results.
[0078] As can be seen from Table 4, the growth trend of FJM60 yeast in Luzhou Laojiao Luzhou-flavor distiller's grains showed a trend of first increasing and then decreasing. Within 48 hours after the initial inoculation, the number of live yeast cells rapidly increased to 2.19×10 8 cfu / mL, indicating that the strain has good growth ability in this environment. However, as time went on, foreign bacteria began to multiply in large numbers, causing the number of live yeast cells to drop significantly after 72 hours and reach its lowest point at 96 hours, accompanied by a noticeable odor, indicating that the fermentation environment has been seriously polluted. Despite this, at 120 hours, the number of live yeast cells rebounded, but was still lower than the initial peak level.
[0079] Compared with Luzhou Laojiao Luzhou-flavor distiller's grains, the growth performance of FJM60 yeast in Wuliangye Luzhou-flavor distiller's grains was more stable (Table 5). Within 48 hours, the number of live yeast cells reached 1.33×10 6 cfu / mL, although lower than the peak value of the Luzhou Laojiao group, the contamination of foreign bacteria was relatively light. As time went on, the number of live yeast cells fluctuated, but generally remained at a certain level, and there was still a certain number of live bacteria at 120 hours. It is worth noting that at 96 hours, despite the contamination of foreign bacteria, the fermentation product still had an aroma, indicating that the strain has a certain ability to resist foreign bacteria in this environment.
[0080] It can be seen from Tables 6, 7 and 8 that after fermentation with FJM60 yeast, the mannan and crude protein contents of various types of liquor lees increased. Among them, the mannan content of Luzhou Laojiao Luzhou-flavor liquor lees reached 0.345% at 48 hours, and the crude protein content increased significantly to more than 15.076%; the mannan content of Wuliangye Luzhou-flavor liquor lees reached 0.542% at 48 hours, and the crude protein content also increased significantly. Sichuan Langjiu Luzhou-flavor liquor lees also showed a similar trend, indicating that FJM60 yeast can effectively promote the transformation and accumulation of nutrients in the substrate during solid-state fermentation.
[0081] In the single yeast fermentation experiment of wheat bran, the addition of 3% urea and 3% ammonium sulfate as nitrogen sources significantly increased the crude protein content of the fermentation product (Table 9). In particular, after fermentation with FJM60 yeast, the crude protein content reached 20.345% and 20.825%, respectively, which was significantly higher than that of the control group without the addition of nitrogen source. At the same time, the dry matter recovery rate was also maintained at a high level, indicating that the strain can effectively utilize nitrogen sources and promote the degradation and conversion of substrates during the fermentation of wheat bran. The increase in true protein content further confirms the potential of FJM60 yeast in improving the nutritional value of feed.
[0082] In summary, FJM60 yeast showed good growth performance and antibacterial ability during solid-state fermentation, and was able to significantly improve the nutrient content of substrates such as distiller's grains and wheat bran. In particular, by adding an appropriate amount of nitrogen source and controlling the fermentation conditions, the quality of the fermentation product can be further optimized and its application value in the feed industry can be improved. Future research can further explore different strain combinations, optimization of fermentation conditions, and deep processing and utilization of fermentation products to promote the widespread application of solid-state fermentation technology in the feed industry.
[0083] Example 3 Evaluation of the nutritional value of fermented feed
[0084] The key nutritional indicators of the fermented wheat bran in Example 2 were measured.
[0085] 1. Index determination method
[0086] Dry matter: Refer to GB / T 6435-2014 Determination of moisture in feed and use a constant temperature air drying oven for determination.
[0087] Crude protein: Refer to GB / T 6432-2018 crude protein determination method in feed, and use FOSS fully automatic Kjeldahl nitrogen analyzer for determination.
[0088] True protein: Determined by copper sulfate method using FOSS fully automatic Kjeldahl nitrogen analyzer.
[0089] Crude fiber: refer to DB37 / T 3370-2018 method for determination of crude fiber content in feed, and use polyester mesh bag-automatic fiber analyzer for determination.
[0090] Energy: measured using an IKA C6000 oxygen bomb calorimeter.
[0091] Reducing sugar: DNS method was used.
[0092] Organic acids: using high performance liquid chromatography.
[0093] 2. Determination of apparent metabolizable energy value of fermented wheat bran in poultry
[0094] Test method: Select Hy-Line brown shell roosters weighing (2.5±0.2) kg to conduct gavage emptying tests on various feed raw materials according to GB / T26437-2010 "Technical regulations for the determination of apparent metabolizable energy of chicken feed by gavage for evaluation of the effectiveness and safety of livestock and poultry feeds". Each raw material was repeated 8 times, and each repeat had 1 chicken. At least one week before the start of the metabolic test, each chicken underwent excrement collection bottle suture surgery and recovered to normal body condition. Each raw material was tested in three stages: pre-feeding period (3d), test period (48h fasting emptying, gavage, 48h collection of excrement) and body condition recovery period. The start time of fasting emptying was accurately recorded, and water was freely available during the fasting period. After the fasting ended, each chicken was accurately gavaged with 25-mesh air-dried feed (preparation method: fermented wheat bran was directly dried in the open air, and the wet material was evenly air-dried) 50g through a gavage, and the completion time of gavage was recorded by individual. All excrement was collected 48 hours after force-feeding, dried at 65℃ and then rehydrated for 24 hours. The gross energy and amino acid content were determined and the apparent metabolizable energy of the feed raw materials was calculated.
[0095] The apparent metabolizable energy (AME, in MJ / kg) of feed for each replicate group of chickens was calculated as follows:
[0096]
[0097] GE1=E1×M1;
[0098] GE2=E2×M2.
[0099] Where:
[0100] GE1——total energy intake, in joules (J);
[0101] GE2——gross excrement energy, in joules (J);
[0102] E1——energy value of dry matter intake, in joules per gram (J / g);
[0103] M1——dry matter intake, in grams (g);
[0104] E2——energy value of dry matter of excrement, in joules per gram (J / g);
[0105] M2——Dry matter mass of excrement 48 hours after ingestion of the tested feed, in grams (g). The results of key indicators of fermented wheat bran (air-dried basis) are shown in Table 10.
[0106] Table 10
[0107]
[0108]
[0109] The apparent metabolizable energy value of fermented wheat bran for laying roosters was (10.8) MJ / kg, which was 27.7% higher than that of unfermented wheat bran (8.53) MJ / kg. In addition, the content of true protein, reducing sugar and organic acid in wheat bran was significantly increased after fermentation. This result fully proves the positive role of FJM60 in improving the nutritional value of wheat bran.
[0110] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, ordinary technicians in the relevant field should understand that the specific implementation methods of the present invention can still be modified or replaced by equivalents. Any modification or equivalent replacement that does not depart from the spirit and scope of the present invention should be covered within the scope of protection of the claims of the present invention.
Claims
1. A yeast that utilizes non-protein nitrogen, characterized in that: The yeast that utilizes non-protein nitrogen is named Candida utilis FJM60; The deposit number of the Candida utilis FJM60 is CGMCC No.31888.
2. An application of the yeast utilizing non-protein nitrogen according to claim 1, characterized in that: The application includes at least one of the following: A1) Application in fermented wheat bran; A2) Application in fermentation of distiller's grains; A3) Application in the preparation of biological feed additives; A4) Application in the preparation of biological feed; A5) Application in degradation of organic pollutants; A6) Application in improving apparent metabolizable energy of poultry.
3. The use according to claim 2, characterized in that: The organic pollutant is wheat bran or distiller's grains.
4. A bacterial agent, characterized in that The bacterial agent includes the Candida utilis FJM60 described in claim 1.
5. A method for preparing bacterial protein feed, characterized in that: include: The bacterial protein feed is obtained by fermenting the fermentation substrate using the Candida utilis FJM60 described in claim 1.
6. The method for preparing bacterial protein feed according to claim 5, characterized in that: The fermentation substrates are wine lees and ammonia water.
7. The method for preparing bacterial protein feed according to claim 5, characterized in that: The fermentation substrates are wheat bran, urea and ammonium sulfate.