Optimisation of culture conditions and development of a novel fed‐batch strategy for high production of β‐galactosidase by Kluyveromyces lactis

The aim of this study was to enhance β‐galactosidase production by Kluyveromyces lactis CICC1773. Firstly, the optimum culture conditions were obtained by response surface methodology, and the maximum β‐galactosidase activity reached 20.6 U mL^?1, about two‐fold increase than that of the initial conditions (initial fermentation medium and conditions). To further improve β‐galactosidase production, a new fed‐batch strategy based on pH feedback control was developed successfully in a 7‐L fermenter, using 400 g L^?1 lactose as feeding medium. As a result, the β‐galactosidase activity and productivity reached up to 111.61 U mL^?1 and 5.31 U/(mL·h), enhanced by 15.3‐fold and 17.6‐fold superior than the results of initial conditions, respectively. To our knowledge, β‐galactosidase activity obtained was the highest value among the results reported by nonrecombinant strains. These results demonstrated that the new fed‐batch strategy based on optimum culture conditions could be automatic control easily and be conductive to further scale up for industrial fermentation.     

Development of efficient enzymatic production of theanine by γ‐glutamyltranspeptidase from a newly isolated strain of Bacillus subtilis,SK11.004

BACKGROUND: Theanine, a unique amino acid found almost exclusively in tea plants, has various favourable physiological and pharmacological functions in humans. Gamma‐glutamyltranspeptidase (GGT, EC 2.3.2.2) is considered to be the most effective enzyme for the production of theanine. In fact, GGT can catalyse the transfer of γ‐glutamyl moieties from γ‐glutamyl compounds to water (hydrolysis) or to amino acids and peptides (transpeptidation). RESULTS: A novel strain, SK11.004, which produces GGT with high theanine‐forming ability was isolated from fermented shrimp paste and identified as Bacillus subtilis through its physiological and biochemical properties as well as its 16S rDNA sequence analysis. Theanine (18.9 mmol L^?1) was synthesised by GGT (0.06 U mL^?1) through transfer reaction in the presence of glutamine (20 mmol L^?1) as a donor and ethylamine HCl (50 mmol L^?1) as an acceptor at pH 10 and 37 °C for 4 h, the conversion rate being up to 94%. CONCLUSION: The enzymatic synthesis of theanine using GGT from a newly isolated strain Bacillus subtilis SK11.004 was found to be an efficient method. Moreover, compared with others, the GGT from B. subtilis SK11.004 exhibited the highest ratio of transferring activity to hydrolytic activity using glutamine, suggesting a high potential application in the production of theanine and other functional γ‐glutamyl compounds. Copyright © 2010 Society of Chemical Industry     

Bioactive action of β‐glucosidase enzyme of Bifidobacterium longum upon isoflavone glucosides present in soymilk

Bioconversion of isoflavone glucosides and antioxidant activity by probiotic strain (Bifidobacterium longum) during soymilk fermentation was investigated, as well as partial characterisation of the produced enzyme β‐glucosidase. The enzyme has higher affinity for genistin than for other substrates assayed. Maximum activity occurred at 42 °C and at pH 6.0; keeping 70–80% of activity for 60 days stored at low temperatures. Bifidobacterium longum grew well in soymilk (8.26 log CFU mL^?1 and pH of 3.9 at 24 h) and were produced in good quantities of organic acids. High hydrolysis degree of isoflavone glucosides (81.2%) was observed at 24 h. Enhancements in bioactivity were assessed in fermented soymilk by monitoring the radical‐scavenging activity, antioxidant activity and DNA protective action. The use of probiotic Bifidobacterium strain as β‐glucosidase producer increased bioactive isoflavone content and demonstrated that this enzyme plays a key role in the bioavailability of soymilk isoflavones, reducing the bioconversion time compared to other studies.     

Characterization of an α‐amylase from sorghum (Sorghum bicolor) obtained under optimized conditions

Sorghum malt α‐amylase can compete with bacterial α‐amylase in industrial applications, if sufficiently stable and produced in a large enough quantity. Conditions for maximal α‐amylase production in sorghum malt and the physico‐chemical properties of the α‐amylase so produced are reported in this study. Sorghum grains were steeped in buffers with varying pH (4.0–8.0) for 24 h, at room temperature, and germinated for another 48 h to obtain the green malt. The buffer that induced the highest quantity of α‐amylase was chosen as the optimal pH and served as the medium for further steeping experiments conducted at different temperatures (10, 20, 30, 40, 50 and 60°C). The α‐amylase activity in the extract was determined in order to obtain the optimum temperature for α‐amylase induction at this particular pH. For the purpose of comparison, the α‐amylase produced at the optimum pH and temperature was purified to apparent homogeneity by a combination of ion‐exchange and size‐exclusion chromatography, and further characterized. Eight‐fold higher α‐amylase activity was induced in pH 6.5 buffer at 20°C compared with water, the traditional steeping medium. The K_m and V_max were estimated to be 1.092 ± 0.05 mg mL^?1 and 3516 ± 1.981 units min^?1, respectively. The activation energy of the purified amylase for starch hydrolysis was 6.2 kcal K^?1 mol^?1. Chlorides of calcium and manganese served as good activators, whereas CuSO₄ inhibited the enzyme with a 42% loss in activity at 312 mm salt concentration. Copyright © 2012 The Institute of Brewing & Distilling     

In vitro inhibitory effects of cranberry bean (Phaseolus vulgaris L.) extracts on aldose reductase, α‐glucosidase and α‐amylase

The in vitro inhibitory activities of different seed extracts prepared from cranberry bean mutant SA‐05 and its wild‐type variety Hwachia against aldose reductase, α‐glucosidase and α‐amylase were examined. The results indicated that the polyphenolics‐rich extracts obtained using 800 g kg^?1 methanol and 500 g kg^?1 ethanol demonstrated inhibitory activities against aldose reductase (IC₅₀ of 0.36–0.46 mg mL^?1) and α‐glucosidase (IC₅₀ of 1.32–1.94 mg mL^?1). The 500 g kg^?1 ethanol extracts also showed α‐amylase inhibitory activities (IC₅₀ of 70.11–80.22 μg mL^?1). Subsequent extracts, prepared further with NaCl and H₂O from precipitates of 800 g kg^?1 methanol or 500 g kg^?1 ethanol extracts, exhibited potent α‐amylase inhibitory activities (IC₅₀ of 17.68–38.68 μg mL^?1). A combination of 500 g kg^?1 ethanol extraction plus a subsequent H₂O extraction produced highest polyphenolics and α‐amylase inhibitors. The SA‐05 α‐amylase inhibitor extracts showed greater inhibitory activities than that of Hwachia. Thus, cranberry bean mutant SA‐05 is an advantageous choice for producing anti‐hyperglycaemic compounds.     

Phytosterols in banana (Musa spp.) flower inhibit α‐glucosidase and α‐amylase hydrolysations and glycation reaction

Three phytosterols were isolated from Musa spp. flowers for evaluating their capabilities in inhibiting glucosidase and amylase activities and glycation of protein and sugar. The three phytosterols were identified as β‐sitosterol (PS1), 31‐norcyclolaudenone (PS2) and (24R)‐4α, 14α, 4‐trimethyl‐5α‐cholesta‐8, 25(27)‐dien‐3β‐ol (PS3). IC₅₀ values (the concentration of inhibiting 50% of enzyme activity) of PS1, PS2 and PS3 against α‐glucosidase were 283.67, 11.33 and 43.10 μg mL^?1, respectively. For inhibition of α‐amylase, the IC₅₀ values of PS1, PS2 and PS3 were 52.55, 76.25 and 532.02 μg mL^?1, respectively. PS1 was an uncompetitive inhibitor against α‐amylase with K_m at 5.51 μg mL^?1, while PS2 and PS3 exhibited a mixed‐type inhibition with K_m at 52.36 and 2.49 μg mL^?1, respectively. PS1 and PS2 also significantly inhibited the formation of advanced glycation end products (AGEs) in a BSA–fructose model. The results suggest that banana flower could possess the capability in prevention of the diseases associated with abnormal blood sugar and AGEs levels, such as diabetes.     

Changes of porcine pancreas α‐amylase in activity and secondary conformations under inhibition of tea polyphenols

To investigate two‐sided functions of tea polyphenols (TP) in antinutrition and energy balance modulation, TP were extracted from Chinese green tea and used to complex porcine pancreas α‐amylase (PPA). Changes of PPA in activity and secondary conformations were analysed. Porcine pancreas α‐amylase was found sensitive to TP treatment. Tea polyphenols exhibited IC₅₀ at 0.41 mg mL^?1 against PPA and maximum inhibitory rate (98.17%) at 3.0 mg mL^?1. Tea polyphenols inhibition was concluded as noncompetitive pattern based on its unchanged Km value (0.98 mg mL^?1) for soluble starch substrate. Tea polyphenols inhibition arose from pH 1.5 to 10.14, covering gastric and intestinal environments inside body. Circular dichroism spectra analysis revealed regular changes of PPA in secondary conformations (increased proportions of α‐helix and β‐sheet) prior to its inactivation at low TP concentrations. Tea polyphenols‐inhibited PPA had distinct double‐negative peaks at 204 nm and 208 nm. Porcine pancreas α‐amylase was inactivated by TP in ways of complexation and modification of secondary conformations.     

Production of a Lactobacillus plantarum starter with linamarase and amylase activities for cassava fermentation

Lactobacillus plantarum strain A6 isolated from cassava, cultured on cellobiose MRS medium showed a growth rate of 0.41 h^?1, a biomass yield of 0.22 g g^?1, and produced simultaneously an intracellular linamarase (76.4 U g^?1 of biomass) and an extracellular amylase (36 U ml^?1). The synthesis of both enzymes was repressed by glucose. The use of such a strain as a cassava fermentation starter for gari production had the following influences: a change from a hetero-fermentative pattern observed in natural fermentation to a homofermentation, a lower final pH, a faster pH decline rate and a greater production of lactic acid (50 g kg^?1 DM). However, this starter did not appear to play a significant role in cassava detoxification, since it was observed that the level of endogenous linamarase released during the grating of the roots was sufficient to permit the complete and rapid breakdown of linamarin.     

Antioxidative and anti‐inflammatory potential of phenolics from purple basil (Ocimum basilicum L.) leaves induced by jasmonic,arachidonic and β‐aminobutyric acid elicitation

The study presents changes in the phenolic levels, antioxidant and anti‐inflammatory potential of purple basil leaves caused by different chemical elicitors: arachidonic acid (AA), jasmonic acid (JA) and β‐aminobutyric acid (BABA). The application of the all tested elicitors increased the concentration of phenolic compounds, including flavonoids and phenolic acids; especially, in comparison with control (457.62 μg g^?1 FW), the rosmarinic acid level significantly increased after AA and JA treatment ‐ 705.0 and 596.5 μg g^?1 FW, respectively. Phenolics from AA‐elicited plants showed the highest anti‐inflammatory activities designated as lipoxygenase (EC₅₀ = 1.67 mg FW mL^?1) and cyclooxygenase inhibition (EC₅₀ = 0.31 mg FW mL^?1). Elicitors' treatments (especially AA and JA) may be a very useful biochemical tool for improving the production of phenolic compounds in purple basil leaves.     

Improvement of L(+)‐lactic acid production from cassava wastewater by Lactobacillus rhamnosus B 103

BACKGROUND: L (+)‐Lactic acid is used in the pharmaceutical, textile and food industries as well as in the synthesis of biodegradable plastics. The aim of this study was to investigate the effects of different medium components added in cassava wastewater for the production of L (+)‐lactic acid by Lactobacillus rhamnosus B 103. RESULTS: The use of cassava wastewater (50 g L^?1 of reducing sugar) with Tween 80 and corn steep liquor, at concentrations (v/v) of 1.27 mL L^?1 and 65.4 mL L^?1 respectively led to a lactic acid concentration of 41.65 g L^?1 after 48 h of fermentation. The maximum lactic acid concentration produced in the reactor after 36 h of fermentation was 39.00 g L^?1 using the same medium, but the pH was controlled by addition of 10 mol L^?1 NaOH. CONCLUSION: The use of cassava wastewater for cultivation of L. rhamnosus is feasible, with a considerable production of lactic acid. Furthermore, it is an innovative proposal, as no references were found in the scientific literature on the use of this substrate for lactic acid production. Copyright © 2010 Society of Chemical Industry     

Effect of gums on viability and β‐galactosidase activity of Lactobacillus spp. in milk drink during refrigerated storage

The viability and β‐galactosidase activity of four Lactobacillus strains in milk drink containing gums during 28 days of refrigerated storage at 4 °C were assessed. The population of Lactobacillus rhamnosus GGB101 and Lactobacillus rhamnosus GGB103 were maintained, whereas the population of Lactobacillus reuteri DSM20016 and Lactobacillus reuteri SD2112 significantly decreased. The recommended level of 6 log CFU g^?1 was exceeded for all tested trains throughout storage. The highest viable number of Lactobacillus rhamnosus GGB103 (8.76 ± 0.03 log CFU mL^?1) was obtained in the product containing carrageenan–maltodextrin. The addition of guar–locust bean–carrageenan led to 20‐fold increase in the level of β‐galactosidase activity for L. rhamnosus GGB101 (1208 ± 2.12 Miller units mL^?1) compared to the control (61 ± 2.83 Miller units mL^?1). Our results suggested that gums could be added to milk to improve viability and enhance β‐galactosidase activity of Lactobacillus.     

Physical,proximate, functional and pasting properties of four non‐ and γ‐irradiated bambara groundnut (Vigna subterranean) cultivars

Effect of γ‐irradiation (2–30 kGy) on physical, proximate, functional and pasting properties of four bambara groundnut cultivars was investigated. Packed (0.74–0.75 g mL^?1) and loose (0.71–0.76 g mL^?1) density varied significantly among cultivars. Generally, CIE L and hue angle decreased, while CIE a, b, deltachroma and colour intensity increased with increased dose. Protein (21.23–23.77 g/100 g), fat (6.38–7.69 g/100 g), crude fibre (1.28–3.54 g/100 g), ash (1.50–4.50 g/100 g) and moisture (10.67–12.92 g/100 g) of non‐ and γ‐irradiated bambara varied with cultivars and dose. Loose (0.41–0.49 g mL^?1) and packed (0.54–0.74 g mL^?1) densities, water absorption (1.62–2.38 g/g) and swelling (10.50–18.00 g/g) increased marginally, while oil absorption (1.93–2.82 g/g), alkaline water retention (0.66–1.23 g/g), emulsion capacity (40.31–58.23%) and stability (31.67–46.49%) decreased with increased dose. Foam capacity (6.85–22.37%) and stability (1.80–22.37%) of γ‐irradiated flours were higher than their nonirradiated (2.03–18.26%) counterparts. Nonirradiated flours showed significantly higher viscosities than their γ‐irradiated counterparts. Flours showed slightly shear‐thinning behaviours.     

Optimisation of lipase production by a mutant of Candida antarctica DSM‐3855 using response surface methodology

Response surface methodology (RSM) was used to determine the optimum cultivation conditions for lipase production by a mutant strain of Candida antarctica DSM‐3855. Among four variables, initial pH, soybean meal, and temperature were identified as significant variables for lipase production by full factorial design. A mathematical model was developed to investigate the influences of three factors on lipase activity and to predict their optimum value. According to RSM, the optimum cultivation conditions for the maximum lipase production were found to be: initial pH 6.0, soybean meal 3.38% (w/v), and temperature 26 °C. Under the optimum conditions, the maximum lipase yields of 27.34 U mL^?1 was obtained after 58 h of cultivation in a 15 L fermenter.     

Purification and Some Properties of a Protease from Sorghum Malt Variety KSV8–11

A protease from sorghum malt variety KSV8–11 was purified by a combination of dialysis against 4 M sucrose, ion‐exchange chromatography on Q‐Sepharose (Fast flow), gel filtration chromatography on Sephadex G‐100 and hydrophobic interaction chromatography on Phenyl Sepharose CL‐4B. The enzyme was purified 5‐fold to give a 14.1% yield relative to the total activity in the crude extract and a final specific activity of 1348.9 U mg^?1 protein. SDS‐PAGE revealed a single migrating protein band corresponding to a relative molecular mass of 16 KDa. Using casein as substrate, the purified protease had optimal activity at 50°C and maximal temperature stability between 30°C and 40°C but retained over 64% of its original activity after incubation at 60°C for 30 min. The pH optimum was 5.0 with maximum stability at pH 6.0 but 60% of the activity remained after 24 h between pH 5.0 and 8.0. The protease was inhibited by Ag⁺, Ca²⁺, Co²⁺, Fe²⁺, Mg²⁺, iodoacetic acid (IAA) and p‐chloromercuribenzoate (p‐CMB), stimulated by Cu²⁺, Sr²⁺, phenylmethylsulfonyl‐fluoride (PMSF) and 2‐mercaptoethanol (2‐ME) while Mn²⁺ and ethylenediaminetetraacetic acid (EDTA) had no effect. The purified enzyme had a K_m of 18 mg·mL^?1 and a V_max of 11.1 μmol · mL^?1 · min^?1 with casein as substrate.     

Production of α‐amylase by Aspergillus oryzae As 3951 in solid state fermentation using spent brewing grains as substrate

BACKGROUND: The optimisation of nutrient levels for the production of α‐amylase by Aspergillus oryzae As 3951 in solid state fermentation (SSF) with spent brewing grains (SBG), an inexpensive substrate and solid support, was carried out using response surface methodology (RSM) based on Plackett–Burman design (PBD) and Box–Behnken design (BBD). RESULTS: In the first optimisation step a PBD was used to evaluate the influences of related factors. Corn steep liquor, CaCl₂ and MgSO₄ were found to be the most compatible supplements to the substrate of SBG and influenced α‐amylase activity positively. In the second step the concentrations of these three nutrients were optimised using a BBD. The final concentrations (g/g dry substrate basis) in the medium optimised with RSM were 1.8% corn steep liquor, 0.22% CaCl₂ and 0.2% MgSO₄ · 7H₂O using SBG as the solid substrate. The average α‐amylase activity reached 6186 U g^?1 dry substrate under the optimised conditions at 30 °C after 96 h. Under the optimised conditions of SSF an approximately 17.5% increase in enzyme yield was observed. CONCLUSION: SBG was found to be a good substrate for the production of α‐amylase by A. oryzae As 3951 under SSF. Copyright © 2007 Society of Chemical Industry     

Bioconversion of shrimp shell waste for the production of antioxidant and chitosan used as fruit juice clarifier

The pH, proteolytic activity, extent of demineralisation and deprotenisation of shrimp waste were studied during 7 days of fermentation using Pseudomonas aeruginosa A2. After 3 days, pH dropped from 7.0 to 4.4 and then remained constant. Simultaneously, a demineralisation of 92% was achieved. However, protease activity reached its highest level (1230 U mL^?1) after 1 day of incubation, and a protein removal of 90% was achieved. Chitin obtained was converted to chitosan. This chitosan, with 73% deacetylation, was tested for clarification of different fruit juices. It was observed that low concentrations of chitosan (below to 1%) greatly increase the clarity of juices without affecting the nutritional value. The antioxidant activity of the hydrolysates produced during fermentation was tested. Hydrolysate obtained after 3 days showed the strongest scavenging activity (90%), which was comparable to the positive control BHA; however, that obtained after 1 day exhibited the highest ferric‐reducing antioxidant power (OD 700 nm = 1.7).     

Quality evaluation of Korean soy sauce fermented in Korean earthenware (Onggi) with different glazes

The aim of this study was to investigate the effect of porosity‐controlled earthenware as fermentation vessels for Korean soy sauce. Porosity of fermentation vessels was controlled by glazing the surfaces of Korean earthenware. Three kinds of onggis– the outside glazed, inside and outside glazed, and unglazed onggi – were made and used to investigate the effect of glazing on the fermentation of soy sauce. During fermentation of soy sauce in porosity‐controlled earthenwares at 30 °C for 4 months, physical, chemical, microbiological and sensory quality attributes were monitored. Compared to other vessels, soy sauce fermented in onggi with both inside and outside surfaces glazed showed less water loss (10.7%), salt content (17.6%) and pH (pH4.4) after the fourth month. It also produced higher total acidity (1.43%), protease activity (810 μg mL^?1 min^?1) and microbiological changes that included total aerobic bacteria [4.3 log(cfu mL^?1)], lactic acid bacteria [3.8 log(cfu mL^?1)] and yeast [4.2 log(cfu mL^?1)]. The contents of total nucleotide (200–255 mg per 100 g sample) and free amino acids (4634–4848 mg per 100 g sample) in soy sauce were not consistent with glazing, which may be more affected by other factors, such as water loss, than the porosity of vessels. However, the percentage of glutamic acid among total free amino acids was 23.6% in onggi with both surfaces glazed, which was a little higher than the 21.9% in the outside glazed and 21.5% in the unglazed. These positive physicochemical and microbiological changes during fermentation in onggi with both sides glazed also resulted in higher sensory quality.     

Production,partial purification and properties of β‐mannanases obtained by solid substrate fermentation of spent soluble coffee wastes and copra paste using Aspergillus oryzae and Aspergillus niger

In order to achieve a higher added value of two galactomannan‐containing wastes, copra paste and spent coffee from the soluble coffee industry (SCW), solid substrate fermentation (SSF) was used. Filamentous fungi Aspergillus oryzae and A niger were used to evaluate the feasibility of producing β‐mannanase by SSF. A 2³ factorial design was used to select the best interaction among the two fungi, the two substrates and two fermentation times. The treatment ‘A niger–copra–2.5 days’ produced a significantly higher (p < 0.05) β‐mannanase activity, having five different isoforms of the enzyme, one of which was partially purified to a specific activity of 764 U mg⁻¹ (U = nmol of mannose released per second from a galactomannan substrate). Copra paste had a higher mannose/galactose ratio (14:1) than SCW (6:1), and low oil content, which led to higher β‐mannanase production from SSF. A β‐mannanase from SSF of copra produced by A oryzae was highly purified using acetone precipitation and cation exchange and size exclusion chromatographies. This enzyme had an MW of 110 kDa, a pI between 3.5 and 4.5 and a specific activity of 1760 U mg⁻¹; purification achieved was 90.7 times. The temperature and pH for optimal activity were 40 °C and 6.0 respectively. The optimal temperature was lower and the optimal pH higher than others previously reported (produced by submerged fermentation), which could be important for viscosity reduction of concentrated coffee extract in instant coffee manufacture. Copra is an interesting alternative for β‐mannanase production, since it is readily available in Mexico; moreover, the residue after SSF has a reduced galactomannan content and may be used for monogastric animal feed. © 2000 Society of Chemical Industry     

Comparison of the performances of different fermentation strategies on cell growth and bacteriocin production by Lactobacillus curvatus CWBI‐B28

The dynamics of cell growth and bacteriocin production by Lactobacillus curvatus CWBI‐B28 in modified De Man/Rogosa/Sharp (mMRS) broth with various concentrations of glucose and complex nitrogen source (CNS; peptone, yeast extract and meat extract) was investigated in flask fermentations and in a laboratory fermentor using batch and fed‐batch cultivations. In fed‐batch fermentation the rate of feeding of the reactor with the substrates was either maintained constant (0.12 L h^?1) or varied exponentially as a function of time. The results showed that both cell growth and bacteriocin activity were influenced by changes in the concentrations of glucose and CNS. Optimal growth and bacteriocin activity were obtained in mMRS broth containing 40 g L^?1 glucose and 40 g L^?1 CNS (mMRS_40/40). A bacteriocin titre of 4266 AU mL^?1 and a cell count of 8.7 log colony‐forming units (cfu) mL^?1 were recorded when this medium was used for cultivation. In batch fermentation using the same medium, a higher cell count (9.5 log cfu mL^?1) and twice as much bacteriocin as in flask fermentation were produced. The highest bacteriocin titre (8533 AU mL^?1) was obtained with fed‐batch fermentation at an exponentially varying rate of feeding. Bacteriocin activity and cell dry mass did not always correlate. Copyright © 2007 Society of Chemical Industry     

Inhibitory activity towards human α‐amylase in wheat flour and gluten

Wheat α‐amylase inhibitors (AI) have been targeted as potential triggers of noncoeliac gluten or wheat sensitivity (NCGS). The aim of this study was to determine AI activity towards α‐amylase from human source in wheat cultivars. Contrary to barley, buckwheat, or oats, high level of AI activity was found in wheat and, to a lesser extent, rye. AI activity (mean IC₅₀ = 137 μg mL^?1) did not vary with respect to ancient or recently developed wheat cultivars. Vital wheat gluten had very high and heat‐stable AI activity (mean IC₅₀ = 23 μg mL^?1), higher than wheat starch (?10 000 μg mL^?1) or acarbose (40 μg mL^?1), a medication for the management of hyperglycaemia and potentially causing digestive disorders due to the accumulation of undigested carbohydrates in the intestine. Data suggest that eating raw wheat gluten, flour or dough could pose a health risk.