Solid-state fermentation of corn-soybean meal mixed feed with Bacillus subtilis and Enterococcus faecium for degrading antinutritional factors and enhancing nutritional value

Background: Corn and soybean meal(SBM) are two of the most common feed ingredients used in pig feeds.However, a variety of antinutritional factors(ANFs) present in corn and SBM can interfere with the bioavailability of nutrients and have negative health effects on the pigs. In the present study, two-stage fermentation using Bacillus subtilis followed by Enterococcus faecium was carried out to degrade ANFs and improve the nutritional quality of corn and SBM mixed feed. Furthermore, the microbial composition and in vitro nutrient digestibility of inoculated mixed feed were determined and compared those of the uninoculated controls.Results: During the fermentation process, B. subtilis and lactic acid bacteria(LAB) were the main dominant bacteria in the solid-state fermented inoculated feed, and fermentation produced a large amount of lactic acid(170 mmo L/kg),which resulted in a lower pH(5.0 vs. 6.4) than the fermented uninoculated feed. The amounts of soybean antigenic proteins(β-conglycinin and glycinin) in mixed feed were significantly decreased after first-stage fermentation with B. subtilis. Inoculated mixed feed following two-stage fermentation contained greater concentratioin of crude protein(CP), ash and total phosphorus(P) compared to uninoculated feed, whereas the concentrations of neutral detergent fiber(NDF), hemicellulose and phytate P in fermendted inoculated feed declined(P < 0.05) by 38%, 53%, and 46%,respectively. Notably, the content of trichloroacetic acid soluble protein(TCA-SP), particularly that of small peptides and free amino acids(AA), increased 6.5 fold following two-stage fermentation. There was no difference in the total AA content between fermented inoculated and uninoculated feed. However, aromatic AAs(Phe and Tyr) and Lys in inoculated feed increased, and some polar AAs, including Arg, Asp, and Glu, decreased compared with the uninoculated feed. In vitro dry matter and CP digestibility of inoculated feed improved(P < 0.05) compared with the uninoculated feed.Conclusions: Our results suggest that two-stage fermentation using B. subtilis followed by E. faecium is an effective approach to improve the quality of corn-soybean meal mixed feed.

Effects of molasses on the fermentation characteristics of mixed silage prepared with rice straw, local vegetable by-products and alfalfa in Southeast China

This experiment was conducted to study the effect of molasses on the fermentation characteristics of mixed silage ensiled rice straw and vegetable by-products with alfalfa.Mixture（202 g kg^-1 dry matter（DM））consisting of rice straw,broccoli residue and alfalfa at the ratio of 5：4：1 was ensiled with three experimental treatments：（1）no additives（control）;（2）molasses at 2.5%（M1）;（3）molasses at 5%（M2）on a fresh matter basis of mixture,respectively.All treatments were packed into laboratory-scale silos,and three silos per treatment were sampled on days 1,3,5,14 and 30.The result showed that the p H value of all mixed silages decreased gradually with the time of ensiling except for the control silage,in which a significant increase（P〈0.05）on day 30 occurred.The lactic acid content increased gradually with the time of ensiling and reached the highest value on day 14,and a marked decrease（P〈0.05）was found in the control silage on day 30.The changes of acetic acid content showed similar pattern with lactic acid content.A trace amount of propionic and butyric acid contents were found in the three mixed silages during the fermentation period.Comparing to the control,M1 and M2 treatments improved the fermentation quality of mixed silages as indicated by higher（P〈0.05）lactic acid contents and lower（P〈0.05）p H and ammonia-N contents.The Flieg points also showed that M1 and M2 silages were well preserved,whereas the control silage had a bad quality.Overall,the findings of this study suggested that adding molasses could improve fermentation quality of mixed silage,and M1 was more suitable for practical application.

Effect of Cellulase and Lactic Acid Bacteria on Fermentation Quality and Chemical Composition of Wheat Straw Silage

The object of this study was to determine the effect of cellulase and lactic acid bacteria (LAB) on fermentation quality and chemical composition of wheat straw silage. Silages were prepared using a small-scale fermentation system and the moisture level was adjusted to 60% of fresh matter (FM) with deionized water. Treatments were designed as: control silage without additives, LAB inoculant Lactobacillus casei Z3-1 (1.0 × 106 cfu·g-1 of FM), commercial inoculant L. plantarum FG 1 (1.0 × 106 cfu·g-1 of FM), Z3-1 + cellulase and FG 1 + cellulase. The neutral detergent fiber (NDF), acid detergent fiber (ADF) and crude protein (CP) contents of the wheat straw prior to ensiling were 76.93%, 48.52% and 4.63% of dry matter (DM), respectively. After 30 days of fermentation, the silages treated with LAB and LAB + cellulase had a lower (P < 0.05) pH and higher (P < 0.05) lactic acid content than the control, and the coliform bacteria, yeast and mold were inhibited at the early stage of fermentation. Besides, silages treated with cellulase had lower (P < 0.05) values of ADF and NDF than the control. The results confirmed that the addition of cellulase and LAB contributed to improving the fermentation quality of wheat straw silage.

Cellulase production by Aspergillus unguis in solid state fermentation

Lignocellulosic substrates are a good carbon source and provide rich growth media for a variety of microorganisms which prodLuce industrially important enzymes. Cellulases are a group of hydrolytic enzymes such as filter paperase (FPase), carboxymethyl cellulase(CMCase) andβ-glucosidase-responsible for release of sugars in the bioconversion of the lignocellulosic biomass into a variety of value-added products. This study examined cellulase production by a newly isolated Aspergillus unguis on individual lignocellulosic substrates in solid state fermentation (SSF). The maximum peak production of enzymes varied from one substrate to another, however,based on the next best solid support and local availability of groundnut fodder supported maximum enzyme yields compared with other solid supports used in this study.Groundnut fodder supported significant production of FPase (5.9 FPU/g of substrate), CMCase (1.1 U/g of substrate) andβ-glucosidase activity (6.5 U/g of substrate) in SSF. Considerable secretion of protein (27.0 mg/g of substrate) on groundnut fodder was recorded. Constant increment of protein content in groundnut fodder due to cultivation of A. unguis is an interesting observation and it has implications for the improvement of nutritive value of groundnut fodder for cattle.

Brewing of low-alcoholic drink from corncobs via yeast-cellulase synchronous fermentation process

The present work focuses on the influence of various parameters, i.e., the dosage of cellulase, the inoculum concentration of yeast, the fermentation temperature and the fermentation time, on the alcohol content and sensory evaluation of the low-alcoholic health drink produced from corncob in a yeast-cellulase synchronous fermentation process. The fermentation was performed by inoculating the seed solution (containing corncob powder and yeast) and cellulase into the synchronous saccharification fermentation medium. Single-factor experiments and orthogonal experiments were performed, and the optimal processing conditions were obtained based on the characterizations of alcohol content and sensory evaluation. The results show that the alcohol content and sensory evaluation of the drink can reach 6.1 vol.% and 92, respectively, when the dosage of cellulase, inoculum concentration of yeast, the fermentation temperature and the fermentation time are 15 U/g, 7%, 32℃ and 84 h, respectively.

Production of Protein Feedstuff by Mixed Fermentation of Waste Lees

[ Objective] To produce protein feedstuff by mix fermentation of waste lees. [ Method] Waste lees was fermented by mixture of Aspergillus niger, Trichoderma vinde and Candida tropicalis to produce protein feedstuff. The fermentation conditions were optimized by single factor test and orthogonal test. [ Result] The optimum fermentation conditions of waste lees are as following： raw materials with fineness of 20 meshes composed of waste lees of 750 g/kg, com meal of 50 g/kg and wheat bran of 200 g/kg; pH value of inoculum culture, 5.0; Aspergillus niger： Trichoderma viHde： Candida tropicalis, 1 ： 1 ： 1 ; inoculum size, 12% （V/V） ; fermentation temperature, 30 ℃; and fermentation time, 6 d. After fermentation, the crude protein content, pure protein content, and crude fiber content were increased by 78.97%, 56.29% and 31.60%, respectively. In addition, the fermented waste lees was rich in beneficial live microorganisms and bioactive substances such as enzymes and vitamins. [ Conclusion] Waste lees can be well fermented to produce protein feedstuff by mix fermentation.

Study on the Saccharification Process of Citrus Vinegar by Fruit-Grain Mixed Fermentation

This study aimed to enhance acid production rate and yield of citrus vinegar, and improve the quality of liquid fermented with fruit juice. [ Method] The saccharification process of citrus vinegar was investigated preliminarily with fruit-grain mixed fermentation method using liquor starters as saccharif- erous agents. [Result] The results indicated that the optimal glutinous rice saccharification conditions were as follows： fruit-grain ratio 3： 1, Aspergillus niger- Monascus purpureus ratio 3： 1, saccharifieafion temperature 60℃, and saccharification time 2.5 h. Under the optimized saccharification conditions, reducing sugar content in saceharified glutinous rice reached 146.43 mg/ml; after fermentation, alcohol content in fermentation broth was 6%, amino nitrogen content was 0.44 mg/ml, and acid yield was 3.1 g/L. [ Conclusion] Citrus vinegar brewed based on the optimized technological conditions exhibited better sensory and physico-chemical properties than that brewed with pure juice.

ETHANOL FERMENTATION OF MIXED GLUCOSE AND XYLOSE BY PICHIA STIPITIS

Pichia stipitis CBS 5773 yeast cells were used to ferment the mixed substrates consisted of glucose andxylose to produce ethanol.The effects of aeration rate,initial substrate concentration and pH on substrateutilization and ethanol yield were evaluated.During batch fermentation,the oscillation phenomena in cell growthwere observed at low aeration rate,whereas the diauxic growth at high aeration rate.The substrate utilizationratio and ethanol yield reached 95％ and 0.46g/g respectively under appropriate operation conditions.Amodified unstructural model was proposed to simulate the diauxic cell growth,substrate consumption andproduct formation.

Study on Addition Amount of Lactic Acid Bacteria and Cellulase in Mixed Silage of Potato Plants

[Objectives]This study was conducted to investigate the appropriate addition amounts of lactic acid bacteria and cellulase in the mixed silage of potato plants,so as to provide a basis for the rational use of potato plants.[Methods]Fresh potato(Solanum tuberosum)seedling plants,watermelon(Citrullus lanatus)plants,and melon(Cucumis melo)plants were selected as raw materials for silage,each of which was added at a ratio of 32%,and 4%of brown flour was added additionally.On this basis,an L 9(32)orthogonal experiment was designed to investigate the effects of the amount of lactic acid bacteria(10,20,30 g/kg)and the amount of cellulase(5,10,15 g/kg)on the experimental results with sensory evaluation score as an investigation index.[Results]With the increase of the amount of lactic acid bacteria and the amount of cellulase,the sensory evaluation score of silage increased,and it was the highest when the amount of lactic acid bacteria added was 20 g/kg and the amount of cellulose was 10 g/kg.With the increase of the amounts of lactic acid bacteria and cellulase added,ammonia nitrogen showed a decreasing trend,and the pH and ammonia nitrogen were the lowest when the amount of lactic acid bacteria added was 20 g/kg and the amount of cellulose was 10 g/kg.[Conclusions]This study provides a theoretical basis for the rational use of effective ingredients and nutrients in potato plants and the development of new feed resources.

The Advantages of Methane Production by Combined Fermentation of Lignite and Wheat Straw

Biogasification of coal is important for clean utilization of coal. Experiments on the fermentation of single lignite, single straw and their mixture were performed to explore the variation characteristics of gas production potential, microbial community and methanogenic metabolic pathways of mixture. Research has shown that mixed fermentation of lignite and straw significantly promoted biomethane production. The abundance of hydrolytic acidifying functional bacteria genera (Sphaerochaeta, Lentimicrobium) in mixed fermentation was higher than that in the fermentation of single lignite and single straw. The abundance of some key CAZy metabolic enzyme gene sequences in mixed fermentation group was increased, which was favorable to improve methane production. Aceticlastic methanogenesis was the most critical methanogenic pathway and acetic acid pathway was more competitive in methanogenic mode during peak fermentation. Macrogenomics provided theoretical support for the claim that mixed fermentation of coal and straw promoted biomethane metabolism, which was potentially valuable in expanding methanogenesis from mixed fermentation of lignite with different biomasses.

Influence of lactic acid bacteria, cellulase, cellulase-producing Bacillus pumilus and their combinations on alfalfa silage quality

This study assessed the effects of lactic acid bacteria(LAB), cellulase, cellulase-producing Bacillus pumilus and their combinations on the fermentation characteristics, chemical composition, bacterial community and in vitro digestibility of alfalfa silage. A completely randomized design involving a 8(silage additives)×3 or 2(silage days) factorial arrangement of treatments was adopted in the present study. The 8 silage additive treatments were: untreated alfalfa(control); two commercial additives(GFJ and Chikuso-1); an originally selected LAB(Lactobacillus plantarum a214) isolated from alfalfa silage; a cellulase-producing Bacillus(CB) isolated from fresh alfalfa; cellulase(C); and the combined additives(a214+C and a214+CB). Silage fermentation characteristics, chemical composition and microorganism populations were analysed after 30, 60 and 65 days(60 days followed by exposure to air for five additional days). In vitro digestibility was analysed for 30 and 60 days. Compared with the other treatments, selected LAB a214, a214 combined with either C or CB, and Chikuso-1 had the decreased(P<0.001) pH and increased(P<0.001) lactic acid concentrations during the ensiling process, and there were no differences(P>0.05) among them. Fiber degradation was not significant(P≥0.054) in any C or CB treatments. The a214 treatment showed the highest(P=0.009) in vitro digestibility of dry matter(595.0 g kg–1DM) after ensiling and the highest abundance of Lactobacillus(69.42 and 79.81%, respectively) on days 60 and 65, compared to all of other treatments. Overall, the silage quality of alfalfa was improved with the addition of a214, which indicates its potential as an alfalfa silage inoculant.

Effect of lactic acid bacteria and propionic acid on conservation characteristics,aerobic stability and in vitro gas production kinetics and digestibility of whole-crop corn based total mixed ration silage

This study was conducted to evaluate the effect of lactic acid bacteria and propionic acid on the fermentation quality, aer- obic stability and in vitro gas production kinetics and digestibility of whole-crop corn based totalmixed ration （TMR） silage. Total mixed ration was ensiled with four treatments： （1） no additives （control）; （2） an inoculant （Lactobacillus plantarum） （L）; （3） propionicacid （P）; （4） propionic acid＋lactic acid bacteria （PL）. All treatments were ensiled in laboratory-scale silos for 45 days, and then subjected to an aerobic stability test for12 days. Further, four TMR silages were incubated in vitro with buffered rumen fluid to study in vitro gas production kinetics and digestibility. The results indicated that all TMR silages had good fermentation characteristics with low pH （〈3.80） and ammonia nitrogen （NH3-N） contents, and high lactic acid contents as well as Flieg points. Addition of L further improved TMR silage quality with more lactic acid production. Addition of P and PL decreased lactic acid and NH3-N contents of TMR silage compared to the control （P〈0.05）. After 12 days aerobic exposure, P and PL silages remained stable, but L and the control silages deteriorated as indicated by a reduction in lactic acid and an increase in pH, and numbers of yeast. Compared to the control, addition of L had no effects on TMR silage in terms of 72 h cumulative gas production, in vitro dry matter digestibility, metabolizable energy, net energy for lactation and short chain fatty acids, whereas addition of PL significantly （P〈0.05） increased them. L silage had higher （P〈0.05） in vitro neutral detergent fiber digestibility than the control silage. The results of our study suggested that TMR silage prepared with whole-crop corn can be well preserved with or without additives. Furthermore, the findings of this study suggested that propionic acid is compatible with lactic acid bacteria inoculants, and when used together, although they reduced lactic acid production of TMR silage, they improved aerobic stability and in vitro nutrients digestibility of TMR silage.

Fermentation characteristics of vinegar residue and some natural materials

Solid state fermentation is an attractive process to produce cellulase economically due to its lower financial investment and lower operating costs. Generally available natural materials in our study included vinegar residue, wheat bran, rice bran and wheat straw. Cellulase production was carried out by solid state fermentation using these materials as the substrate of Trichoderma koningii. The ingredients of natural materials, associated with the effects of water content, time and nitrogen sources on cellulase synthesis were studied. A comparatively high cellulase activity （〉4 IU·g^-1 SDM） was obtained in the fermentation batch. The optimum culture time of vinegar residue, wheat bran and wheat straw were 72 hours, but that office bran was 60 hours. Total water contents of wheat bran, rice bran and wheat straw should not exceed 50% and that of vinegar residue should not be more than 60%. It was also shown that nitrogen salts contributed much to fermentation. （NH4）3PO4 and urea achieved good results in promoting enzyme activity.

Production of Citric Acid from Apple Pomace Enzymolyzed by Cellulase

Cellulase can evidently increase the content of glucose and has a significant effect on the production of citric acid from apple pomace by Aspergillus niger. Based on experiments, a cellulolytic enzyme named cellulase A6 was found able to produce about 170 g glucose from 1 kg dried apple pomace after 12 h reaction, with cellulase concentration of 20 U/g in the medium at 50oC, natural pH without pretreatment of alkali. Using the treated apple pomace as a liquid state substrate, Aspergillus niger-C selected out was able to produce about 256 g citric acid from 1 kg dried apple pomace at 35oC in 3 d or 30oC in 5 d with flask rotation speed of 210 r/min, and the conversion of citric acid could reach 80% based on the amount of sugar consumed.

Cellulase Producing Bacteria from the Wood-Yards on Kallai River Bank

This study evaluates the influence of growth parameters such as pH, temperature, Carboxy Methyl Cellulose (CMC) concentration and agitation on cellulase production from three bacterial strains, viz., Achromobacter xylosoxidans BSS4, Bacillus sp. BSS3 and Pseudomonas sp. BSS2 isolated from the wood-yards on Kallai river bank in Kerala. Production of cellulase by these isolates was detected using basal salt medium (BSM) with 0.5% CMC as supplement, and CMCase activity was confirmed by iodine test. Dinitrosalicylic acid method was employed for assaying the cellulase production by measuring the amount of glucose liberated in μmol/mL/min. Maximum enzyme production from Pseudomonas sp. BSS2 was at pH 8, 37℃ with 1% CMC and 150 rpm, and cellulase production increased from initial 49.84 U/mL to 91.28 U/mL after optimization. The highest enzyme activity from Bacillus sp. BSS3 was at pH 9, 37℃ with 1% CMC, 150 rpm, and cellulase production increased from initial 26.05 U/mL to 104.68 U/mL after optimization. The maximum enzyme production from A. xylosoxidans BSS4 was at pH 7, 40℃ with 0.5% CMC and 150 rpm, and cellulase production increased from initial 55.28 U/mL to 68.37 U/mL after optimization. Thus among the three isolates, Bacillus sp. BSS3 showed maximum enzyme yield which can be explored for further scale up studies with an industrial perspective.

Cellulase Production from Species of Fungi and Bacteria from Agricultural Wastes and Its Utilization in Industry: A Review

In energy deficient world, cellulases play a major role for the production of alternative energy resources utilizing lignocellulosic waste materials for bioethanol and biogas production. This study highlights fungal and bacterial strains for the production of cellulases and its industrial applications. Solid State Fermentation (SSF) is more suitable process for cellulase production as compared to submerge fermentation techniques. Fungal cellulosomes system for the production of cellulases is more desirable and resistant to harsh environmental conditions. Trichoderma species are considered as most suitable candidate for cellulase production and utilization in industry as compared to Aspergillus and Humicola species. However, genetically modified strains of Aspergillus have capability to produce cellulase in relatively higher amount. Bacterial cellulase are more resistant to alkaline and thermophile conditions and good candidate in laundries. Cellulases are used in variety of industries such as textile, detergents and laundries, food industry, paper and pulp industry and biofuel production. Thermally stable modified strains of fungi and bacteria are good future prospect for cellulase production.

Cellulase Production by Endophytic Strains of <i>Trichoderma reesei</i>from <i>Baccharis dracunculifolia</i>D. C. (Asteraceae)

Cellulases are enzymes responsible for the degradation of cellulose, the major compound in plant cells. Cellulose is a polysaccharide composed of several glucose units linked together by chemical bonds. Cellulases, such as endoglucanases, beta-glucosidase and exoglucanases, break the chemical bonds between the glucose units. Fungi, including the endophytic species, can be great cellulase producers. This study aimed to evaluate cellulase production by four endophytic strains of Trichoderma reesei semi-solid media containing sugarcane bagasse, supplemented or not with salts. Two fermentations were carried out for 43 days. Samples were taken every seven days to obtain production peaks. The enzymes were characterized by their optimum pH and temperature of activity and stability upon incubation in the presence of ions, pH and temperature variations. The results showed that the endophytic strains FB1, FB2, FB3 and FB4 of Trichoderma reesei produce cellulases in a sugarcane bagasse medium, supplemented or not with salts, at pH 5.5 and 30°C. The supplemented medium proved to be more appropriate to induce cellulase production after 29 days of fermentation, with FB4 having the best yield: 16.32 ± 2.65 IU/gram of fermented substrate.

Production of Cellulase and Xylanase by Aspergillus terreus KJ829487 Using Cassava Peels as Subtrates

Cassava (Manihot esculenta, Crantz) is one of the most important food plants in West Africa. Its peels are made up of cellulose, hemicellulose and lignin. This lignocellulolytic biomass can be converted using microbial enzymes to fermentable sugars which have wide range of biotechnological relevance in many fermentation processes. The aim of this study is to screen filamentous fungi from decaying cassava peels that are good producers of xylanases and cellulases. Decaying parts of cassava peels were obtained and brought to the laboratory for further work. Fungi were isolated, identified and screened for cellulase and xylanase production. Isolate with highest frequency of occurrence and enzyme production was identified using phenotypic and molecular method. Optimisation of growth conditions for enzymes production was monitored using the DNSA method, also saccharification of cassava peel were carried out using the enzymes obtained from the isolate. Aspergillus terreus KJ829487 was the predominant fungus. It produces cellulases and xylanases optimally at 40°C, pH 6 and 8, utilising carboxymethylcellulose (CMC) or xylose and yeast extracts as its carbon and nitrogen sources respectively. Saccharification of the peels yielded 584 mg/L glucose, 78 mg/L xylose and 66 mg/L rhamnose. Aspergillus terreus KJ829487 obtained from cassava peels have the ability to produce high concentration cellulases and xylanases which effectively hydrolysed the lignocelluloses’ biomass to fermentable sugars.

Multi-enzymes Production Studies in Single Tray Solid State Fermentation with Opened and Closed System

The robustness ofA. awamori and A. oryzae as enzyme producers is exploited in fungal fermentation on agricultural solid waste. High-level production of extracellular glucoamylase, protease, cellulase and xylanase has been achieved. Three different types of ＇waste＇ solids （wheat bran, soybean hulls and rapeseed meal） have been used in studies of solid state fermentation （SSF）. The enzymes could be produced in significant levels by continuously supplying oxygen （02） through the tray system known as ＂closed＂ and ＂opened＂ tray systems. A perforated tray system was developed in this study that permits direct access to 02. Testing the tray system with different perforated mesh aperture sizes in this study did not yield different results in growth performance of A. awamori and A. oryzae. A. awamori and A. oryzae can be very versatile in producing various enzymes with different substrates with different starch, protein, hemiceilulose and cellulose contents. These studies indicate that A. awamori is more suitable for the efficient production of multiple enzymes in the closed system including xylanase and cellulase, while the production of glucoamylase and protease is superior in the opened system. A. oryzae is more suitable for the efficient production of protease and cellulase in the closed system, while the production of protease is more favourable the opened system. A. awamori efficiently consumed starch in wheat bran medium and produced very high glucoamylase activity, and after that, the fungus efficiently produced other enzymes to degrade other complex nutrients such as protein, hemicellulose and cellulose. Meanwhile, A. oryzae efficiently consumed protein in rapeseed meal and produced very high protease activity. The ability of both filamentous fungi, to convert biomass through SSF bioconversion will have a great impact on food and agro-industry in every aspect of life from food and medicine to fuel.

Research on Fermentation Technology of Raspberry-mulberry Leaven

The mixed fermentation is based on the raw material of raspberry and mulberry.The research is to explore the sticking point for the fermentation progress.To attain the optimized fermentation condition which gained by integrating fermentation data,the concentration of pectinase should be 80 mg/L,the mass fraction of Lactobacillus,Acetobacter and yeast should be 3%,1%and 3%,respectively,the fermentation temperature should be 28℃,the initial sugar content should be 24°,and the duration of fermentation should be 16 d.After the fermentation,sugar content should be adjusted to 25°,and the products should be stored for 3 d in the condition of 15℃.The Raspberry-mulberry leaven containing 8.5×10^(8) probiotics per milliliter and 61.2 g total acid per liter should be diluted with various proportions of water to drink according to different habits of customers[1].The volume ratio of leaven and water should be at least 1∶1 and at most 1∶3.