Optimization of Medium Composition for Nisin Fermentation with Response Surface Methodology

ABSTRACT:  Nisin is an effective food biopreservative widely used in food industry. However, 1 problem of concern is limited production rate and final nisin concentration. A nisin-producing strain, L. lactis Lac2, a mutant strain with high yield of nisin, was obtained in our laboratory recently. In the present study, a fractional factorial design was applied to investigate the main factors that affect the yield of L. lactis Lac2. Central composite experimental design and response surface methodology were adopted to derive a statistical model for optimizing the composition of the medium. The results showed that the optimum medium for nisin production of L. lactis Lac2 was composed of 2.68% sucrose (w/v), 0.5% tryptone (w/v), 1% yeast extract (w/v), 0.3% Tween-80 (w/v), 0.02% MgSO₄·7H₂O (w/v), 0.81% NaCl (w/v), 1.91% K₂HPO₄ (w/v), 0.05% ascorbic acid (w/v), and 2% agar (w/v) (if necessary) at pH 6.5. When cultured in the optimum medium, the nisin yield is an average of 3381.81 IU/mL, which nearly doubled the yield when incubated in the initial medium. Also, the concentration of tryptone was decreased while that of the sucrose was increased when compared with CM broth, which means a reduction of the fermentation cost.     

Optimization of the fermentation medium for alpha-galactosidase production from Aspergillus foetidus ZU-G1 using response surface methodology

ABSTRACT:  The optimization of fermentation medium for alpha-galactosidase production by Aspergillus foetidus ZU-G1 was investigated in shaker flask fermentation. A one-factor-at-a-time experiment was used to screen the preferable nutriment (carbon sources, nitrogen sources, and essential elements) for alpha-galactosidase production. A fractional factorial design was used to screen the main 5 factors, soybean meal, wheat bran, KH₂PO₄, FeSO₄·7H₂O, and the medium initial pH, that affected the production of alpha-galactosidase. The central composite experimental design was further adopted to derive a statistical model for optimizing the composition of the fermentation medium. The experimental results showed that the optimum fermentation medium for alpha-galactosidase production by Aspergillus foetidus ZU-G1 was composed of 3.2% soybean meal (w/v), 2% wheat bran (w/v), 0.1% KH₂PO₄ (w/v), and 0.05% FeSO₄·7H₂O (w/v); initial medium pH was 6.31. The results further predicted that alpha-galactosidase activity reached 64.75 U/mL after 96-h incubation in this medium, which was approximately 7 times higher than that incubated in the nonoptimized medium. The time course of alpha-galactosidase production in the optimized medium composition was also carried out to validate the model.     

Optimization of beta-carotene production by Rhodotorula glutinis using high hydrostatic pressure and response surface methodology

ABSTRACT:  Rhodotorula glutinis RG6 was treated by high hydrostatic pressure (HHP) of 300 MPa for 15 min for improving its ability of β-carotene production. After the treatments of 5 repeated cycles, the mutant strain RG6p was obtained, β-carotene production of which reached 10.01 mg/L, increased by 57.89% compared with 6.34 mg/L from parent strain RG6. To optimize the medium for β-carotene fermentation by mutant RG6p, a response surface methodology (RSM) approach was used in conjunction with a factorial design and a central composite design, and the maximum yield of β-carotene (13.43 mg/L), an increase of 34.17% compared to the control, was obtained at a pH 6.7 with an optimum medium (40 mL/250 mL) of yeast extract (4.23 g/L), glucose (12.11 g/L), inoculum (30 mL/L), tomato extract (2.5 mL/L), peanut oil (0.5 mL/L), and (NH₄)₂SO₄ (5 g/L).     

Efficacy of Sulfuric Acid Scarification and Disinfectant Treatments in Eliminating Escherichia coli O157: H7 from Alfalfa Seeds Prior to Sprouting

ABSTRACT: E. coli O157:H7 reduction on inoculated alfalfa seeds was investigated using acid scarification treatments with or without subsequent application of sanitizers. Scarification with 0.1 to 2N H₂SO₄ for 2.5 to 45 min did not affect (p ≤ 0.05) seed viability. E. coli O157:H7 was reduced by 2.1 to 5.0 logs after treating with 0.1 to 2N H₂SO₄ for 5 to 20 min. Combined scarification (0.5N H₂SO₄) and H₂O₂ or CH₃COOH treatments enhanced microbial destruction by less than 1 log compared to sanitizer alone. Chlorine, Na₂CO₃, or Na₃PO₄ treatments preceded by scarification did not significantly increase microbial destruction compared to sanitizer alone. Appreciable reductions in seed germination were only observed with chlorine treatments.     

Effect of different extraction and precipitation methods on yield and quality of pectin

An experimental study has been conducted to determine the combined effects of different extraction conditions and precipitation method on the yield and quality of high methoxyl pectin from lemon peels. Pectin was extracted using different mineral acids (HCl, HNO₃ and H₂SO₄) at four concentration levels (0.025; 0.05; 0.1 and 0.2  m ), at 70 °C for 4 h. The soluble pectin was precipitated by iso-propanol or by an aluminium sulphate, Al₂(SO₄)₃, solution at pH 4. The extraction with HCl and HNO₃, at the highest concentrations investigated, followed by aluminium precipitation led to the best results in terms of yield (22–25%), quality and gelling power of pectin with a remarkable decrease of alcohol consumption as compared to the alcoholic precipitation under the same extracting conditions.     

PURIFICATION OF THE ANTI-LISTERIAL BACTERIOCINLIKE INHIBITORY SUBSTANCE PRODUCED BY ENTEROCOCCUS FAECIUM 108

Extracellular antimicrobial substances produced by certain enterococci inhibit Listeria monocytogenes. Enterococcus faecium 108, a competitive food isolate, produced a heat-stable and protease-sensitive anti -L. monocytogenes bacteriocin-like substance (Ef108) in Listeria selective enrichment broth and other media. Ef108 activity was purified to homogeneity by a four-step procedure including (NH₄)₂SO₄ fractionation, chromatography on anion exchange QSepharose column, and two Superose 12 gel filtration columns. In activities represented by two peaks (Ef108A and Ef108B), 90% of the crude activity was due to peak B. Ef108A is believed to be a variable aggregate of the active moiety in Ef108B. All Listeria spp. and five L. monocytogenes serotypes were inhibited by Ef108B in several media including Listeria enrichment medium. The Ef108 activity may be due to a bacteriocin-like inhibitory substance produced by E. faecium 108 that may suppress the growth and predominance of Listeria monocytogenes during selective enrichment .     

IN VITRO COMPARISON OF ANAEROBIC AND AEROBIC GROWTH RESPONSE OF SALMONELLA TYPHIMURIUM TO ZINC ADDITION

Zinc supplemented diets have been used to provide zinc as a nutrient and higher concentrations have been used to induce molt in laying hens. It is not known if the zinc in these diets would inhibit Salmonella spp growth. This study examines the effects of zinc compounds on the growth of S. typhimurium poultry isolate under aerobic and anaerobic conditions. The aerobic growth response of S. typhimurium was determined either in tryptic soy broth (TSB) or minimal (M9) broth containing five different concentrations (0.67, 2.01, 3.35, 4.69, and 6.03% [wt/vol]) of either Zn acetate [Zn(C₂H₂O₂)₂2H₂O] or Zn sulfate [ZnSO₄7H₂O] while anaerobic growth response was determined in M9 broth with or without reductants (L-cysteine hydrochloride [C₃H₇NO₂SHCl], and sodium sulfide [Na₂S 9H₂O]). Aerobic growth rates inhibited (P < 0.05) by Zn acetate than by Zn sulfate in TSB medium. The Zn source and concentration decreased (P < 0.05) aerobic growth response of S. typhimurium poultry isolate in M9 medium. The growth rates of S. typhimurium under anaerobic growth conditions were less responsive to Zn salts but were generally lower (P < 0.05) in the presence of reductant than in the absence of reductants at each concentration of Zn compound. The results in this study provide evidence that Zn may inhibit S. typhimurium under in vitro aerobic or anaerobic atmospheric conditions and S. typhimurium grows less optimally under anaerobic growth conditions.     

AFLATOXIN LIKE FLUORESCENT SUBSTANCES IN SPICES

To minimize the risk of misidentifying some blue fluorescent substances inherent in spices as aflatoxin during analysis, several confirmatory chemical tests and developing solvent systems were evaluated. Chemical confirmation with 20% HCl, 20% H₂SO₄ and TFA and TLC development in chloroform + acetone (95:5 v/v) followed by anhydrous diethyl ether (multiple development) were useful to obviate the interfering fluorescent substances.     

Isolation of Tomato Seed Meal Proteins with Salt Solutions

Salt solutions were used in isolating tomato seed meal proteins. Na₂SO₃ and NaCl solutions at different concentrations, and pH were included in a central composite design to find optimum conditions of protein isolation. The highest total protein yield was achieved with water extraction (no salt present). Salt extraction at pH 7.5 produced isolates with protein content of 93.4% (NaCl 5% w/v) and 77.1% (Na₂SO₃ 0.5% w/v). Observed values were in good agreement with predicted values. Isolates extracted with different salt solutions ranged from less soluble but very resistant to heat and Ca²⁺, to very surface active with functional properties comparable to commercial soy isolates.     

POTENTIAL FOR EXAGGERATION OF PCB CONTENTS OF PACKAGED FISH BY PHTHALATES FROM THE WRAPPING MATERIAL OR SOLVENTS

A convenient method for recovering PCBsfrom lipids is to digest the matrix in concentrated H₂SO₄ and recover the unaffected PCBs into hexane for determination by gas chromatography with electron capture detection. In a recent fish analysis case the peaks for these PCBs were accompanied by a large component identified as di- (2-ethylhexyl) phthalate, shown to have migrated into fish lipidfrom a heavy plastic wrap, and only partly destroyed by the H₂SO₄. The di-n-butyl and di-(2-ethylhexyl) phthalates bracket the major Aroclor peak region of chromatograms on a methyl silicone GC column and possibly a di-n-hexyl phthalate ester would very likely be superimposed directly on the PCBs. In the course of this work it was found that commercially available hexanes of presumed high quality contained the same di-(2-ethylhexyl) phthalate plasticizer in proportions high enough to be of concern in such analyses.     

Reaction of apple pectin with ammonia

Amidated apple pectins were prepared in aqueous and aqueous-alcoholic media, and the factors influencing both ammonolysis and hydrolysis of the ester groups of apple pectin by ammonia were studied.
In aqueous solution, the extent of ammonolysis and hydrolysis was very dependent upon ammonia concentration. In aqueous alcohol, the products were also dependent upon the particular alcohol and its concentration, increasing concentrations suppressing hydrolysis; the overall rate of reaction is much slower than in the absence of alcohol. The product balance between amonolysis and hydrolysis of the ester groups is influenced by the polarity of the medium, and by the concentration of NH⁺₄ and OH⁻.
It was shown that the extents of ammonolysis and hydrolysis of the ester groups are significantly affected by the polarity of the medium and the acid-base equilibrium of the interaction of ammonia with water, i.e. NH₃+H₂O ↔ NH⁺₄+OH⁻. The content of free ester and amidated carboxyl groups in the pectin can be regulated by varying the concentration of ammonia, temperature, reaction time and the polarity of the medium. The rate of enzymic hydrolysis of amidated pectins decreases with increasing amide content. A mechanism for the reaction is proposed.     

Production of Hydrogen Peroxide by Lactobacillus delbrueckii subsp. lactis as Influenced by Media Used for Propagation of Cells

ABSTRACT: Cells of Lactobacillus delbrueckii subsp. lactis I were grown in several media to determine the most suitable medium for production of H₂O₂ during refrigerated storage. H₂O₂ was detected from cells that were resuspended in phosphate buffer containing variable amounts of glucose. Cells grown in MRS broth produced more H₂O₂ during refrigerated storage than those grown in LBS pr PTM. Cells grown in LBS produced more H₂O₂ then those grown in PTM. For both MRS and LBS, cells inoculated in phosphate buffer containing 0% glucose produced significantly more H₂O₂ than those containing 1% or 10% glucose. MRS medium appears to be the best medium for growing cells for the production of H₂O₂.     

The preparation of protein hydrolysate from defatted coconut and soybean meals

This study was conducted to determine the effects of temperature, time and concentration of acids on the hydrolysis of coconut and soybean meals. Using 6∼ hydrochloric acid (HCl), the complete hydrolysis of soybean meal was reached after 36 hr at 95°C, while it took only 24 hr to complete the process when 18 N sulphuric acid (H₂SO₂) was used at the same temperature. Coconut protein exhibited some degree of resistance to hydrolysis. Using 10 N HC1 and 18 N H₂SO₂ in two separate tests, it took 48 hr to complete hydrolysis at 95°C. Sulphuric acid caused a considerably greater decomposition of amino acids than HCl during longer periods of reaction with high acid concentration and temperature. Flavour development is a function of the free amino acids released which in turn is a function of acid concentration, reaction time and temperature.     

Solubility of amaranth seed proteins in sodium sulphate and sodium chloride: the main factor in quantitative extraction for analysis

Nitrogen was extracted more efficiently from amaranth seed with 0.04 M Na₂SO₄ (5% w/v) than with either 0.09 M or 0.17 M NaCl (5% or 10% w/v), despite both solutions having the same ionic strength (μ= 1). Solubility of saline soluble proteins (albumin ± globulin) was very poor in either water or 1M NaCl, but increased in 0.4M NaCl at alkaline pH between 7 and 10. Globulins were very soluble in 0.4M NaCl at a pH 9. Albumin was the main storage protein. Saline soluble proteins formed very weak gels.     

New oil-gelling agents for cosmetics: formation mechanism of oil gels

New oil-gelling agents have been developed composed of a long-chain dialkyl phosphate (DP) surfactant and aluminium ion or multinuclear aluminum ion (MAI) which provide good oil-gel systems. One such gelling agent is the aluminum salt of DP (DP-Al) prepared as a precipitate by mixing DP with aluminum chloride in aqueous solutions. Addition of small amounts of the salt to non-polar oils led to hardening of the oil solutions at temperature below the melting point of the alkyl chain (Tc) of DP-Al. Results obtained by X-ray diffraction techniques and with scanning electron microscopy (SEM) revealed that linearly-polymerized assemblies of DP-Al expand in oils three-dimensionally, which suggests that the excellent stability of the gel systems arises from the highly ordered structure. The gelling agent of DP-Al offered the ideal rheological property to waxy cosmetic products such as lipsticks. The complexes of DP with MAI particles (1 nm diameter) of aluminum chlorohydrate, Al₁₃O₄(OH)₂₄(H₂O)₁₂CI₇, provided the ideal thixotropic behaviour in non-polar oils. The DP-MAI particle complexes were found to interact weakly by cohesive forces which makes a highly ordered structure of the DP-MAI particle complexes. The DP-MAI particle complexes gave excellent stability and transparency to cosmetic products such as w/o creams.     

DETERMINATION OF CARBOXYMETHYLCELLULOSE IN FOOD PRODUCTS

SUMMARY –A method was developed to determine sodium carboxymethylcellulose (CMC) in food products containing three or more hydrocolloids after prior removal of interfering substances. Papain was used to digest the proteins; calcium chloride was added to precipitate algin and pectic acid and sulfated hydrocolloids were then precipitated by cetylpyridinium chloride (CPC) in the presence of 0.5M NaCl. Celite 535 or Hyflo Super Cel was added, the mixture filtered over Reeve angel No. 202 paper and the residue washed with 0.01% CPC-0.01M NaCl. The filtrate and washings were collected and diluted with distilled water to attain a final concentration of 0.2M NaCl at which concentration the CMC was precipitated selectively by CPC. The mixture was filtered over a Celite 535 or Hyflo Super Cel column and the residue washed with 0.5M Na₂SO₄ and then with 0.01% CPC-0.01M NaCl until the washings were negative to the phenol-H₂SO₄ reagent. Finally, the residue on the column was washed with hot 30% H₂SO₄ and the hydrolyzed CMC in the eluate determined by the 2,7-naphthalenediol test. Based on the amount added to mixtures, recoveries of 75–86% were obtained from milk and other highly complex and proteinaceous products, with a standard deviation of ± 0.58 mg, when 10 mg of CMC were added. The determination is critically dependent upon the degree of substitution (DS) of the CMC. As a consequence, the method cannot be applied with absolute certainty to unknown samples.     

The Binding of H2O2-Generated Fe(III) to Bifidobacterium thermophilum Depends on the Method of its Production

ABSTRACT: Fe(OH)₃ was produced from Fe(II) and H₂O₂ by methods associated with high and low free radical activity. More Fe(OH)₃ became bound to Bifidobacterium thermophilum when the former type of method was used, and protoplasting experiments indicated that binding to both the cell walls and membranes was enhanced. Binding to cell walls was instantaneous, whilst that by the membranes increased with time using both methods. Al(III), a lipid peroxidation enhancer, increased Fe(OH)₃ binding by membranes only. It was concluded that B. thermophilum is able to abate increased activity of free radicals not only by scavenging them, but also by removing iron from their environment.     

Quantitative Determination of Carrageenan in Milk and Milk Products Using Papain and Cetyl Pyridinium Chloride

SUMMARY– Proteins in milk, chocolate milk, evaporated milk, and ice cream containing added carrageenan were digested with papain at 70°C in the presence of 1.0 M NaCl. The digest was adjusted to pH 8.0 to 8.5 with NaOH. Celite was added and the mixture filtered over glass wool. Carrageenan in the filtrate was precipitated with cetyl pyridinium chloride (C.P.C.) in the presence of 0.5 to 1.0 M KCl and Celite. The carrageenan-C.P. precipitate was washed with 0.1% C.P.C.-0.05 M KCI until the filtrate was negative to the Benedict's test. Then, it was dissolved in 30% H₂SO₄ and the carbohydrate content determined by the phenol-H₂SO₄ method.
At concentrations of 0.01 to 0.2% carrageenan, average recoveries of 92 to 102% were obtained from milk. For chocolate milk, evaporated milk and ice cream, and at a level of 0.1% carrageenan, recoveries of 90, 94 and 96%, respectively, were obtained. Optimum conditions for the isolation of the carrageenan cetyl pyridinium complex were established.     

Modulation of Lipid Metabolism by Centella Asiatica in Oxidative Stress Rats

ABSTRACT:  A study was carried out to investigate the effects of Centella asiatica leaf on lipid metabolism of oxidative stress rats. The rats were fed 0.1% hydrogen peroxide (H₂O₂) with either 0.3% (w/w) C. asiatica extract, 5% C. asiatica powder (w/w), or 0.3% (w/w) α-tocopherol for 25 wk. Results of the study showed that C. asiatica powder significantly ( P < 0.05) lowered serum low-density lipoprotein compared to that of control rats (rats fed H₂O₂ only). At the end of the study C. asiatica -fed rats were also found to have significantly ( P < 0.05) higher high-density lipoprotein and lower triglyceride level compared to rats fed only normal diet. However, cholesterol level of rats fed both C. asiatica extract and powder was found to be significantly ( P < 0.05) higher compared to that of control rats. It was interesting to note that consumption of C. asiatica significantly decreased body and liver weights of the rats. Histological examinations revealed no obvious changes in all rats studied. Quantitative analysis of C. asiatica leaf revealed high concentration of total phenolic compounds, in particular, catechin, quercetin, and rutin.     

Comparison of six methylation methods for analysis of the fatty acid composition of albacore lipid

Six methods widely used to produce methyl esters for the gas chromatographic determination of the fatty acid composition of a marine lipid were compared. Four acid-catalyzed methods (1% H₂SO₄: CH₃OH; 5% HCl: CH₃OH; 7% and 14% BF₃: CH₃OH) and two base-catalyzed methods [0.5M NaOCH₃: CH₃OH; (1:4) tetramethylguanidine: CH₃OH] were used.
The use of BF₃: CH₃OH (7% and 14%) gave a lower content of 18:1 n9 than the other methods and produced an artefact (2.7–3.2% of total fatty acid content) eluting between the 20:5 and 24:1 fatty acid methyl esters. No significant differences were obtained between the other four methods.
Accordingly the use of BF₃: CH₃OH for transmethylation of marine lipids is not recommended. Results obtained in the other four methods showed that all are comparable.