A wealthy gambler has put £600,000 on a no vote in the Scottish independence referendum, in what is thought to be the largest political bet in modern times.

The bookies William Hill said a punter who first placed £400,000 on a no vote in June has now increased his stake to £600,000 in the wake of Alex Salmond's below par performance against no campaign leader Alistair Darling in Tuesday's televised referendum debate.

The initial bet by the middle aged customer at a branch in Surrey, described as a businessman who will not have a vote in September's referendum, was close to the record political bet of A$750,000 (£419,000) placed on Australia's general election in September 2013.

William Hill said it believed the increased wager had now made the bet a world record figure, but it was now offering a lower return on the extra £200,000 after cutting the odds on a no vote in September.

The punter got odds of 1/4 on a no vote in June, but William Hill cut the odds since then to 1/6. He stands to make a profit of £133,333. Gamblers backing a yes vote are now being offered longer odds of 9/2, out from 7/2, the company said.

Last week, it emerged that Ralph Topping, the outgoing chief executive of the bookmakers William Hill, who is credited with returning the firm to the FTSE100 had endorsed a yes vote.

Topping said in a statement released by pro-independence group Yes Scotland that he believed there would be a currency union despite pledges from the chancellor, George Osborne, the chief secretary to the Treasury, Danny Alexander, and the shadow chancellor, Ed Balls, that a sterling pact would be vetoed.

"It is not the location of a bank's brass plate in Scotland, but rather the concentration of economic assets in the rest of the UK that matters to British financial stability," Topping said. "The Treasury should stop playing politics and listen to market voices wanting a collaborative approach to the monetary system between an independent Scotland and the rest of the UK. In this respect, common sense economics was always going to trump politics."

Graham Sharpe, a spokesman for William Hill, said: "As the odds about a no vote had lengthened in the run-in to the recent TV debate in anticipation of a mind-changing performance by Alex Salmond, only for Alastair Darling to outshine him, it seems probable that the customer decided that now was the best moment to get the longest odds available, so he added 50% to what was already a record-busting bet."

The Political Betting website reported that the odds on the yes campaign winning the referendum had lengthened further since Tuesday's debate, taking the chances of the yes vote from between 20% to 24% down to 13.5% on Thursday.

Prof David Bell, an economist at Stirling university and an expert in the economics of Scottish independence, said last month that his analysis of the pattern of political betting on the referendum put the odds of a no vote in September at 79%.

Bell said the odds against a yes vote had lengthened partly because of the £400,000 bet taken by William Hill in June. While there was a large number of bets on yes winning, the sums involved were much smaller than the bets placed on no winning.

"Some of the change in odds will also be a response to punters who are willing to bet heavily on a no outcome, including the individual reported widely in the media recently for placing a bet of £400,000 on Scotland staying part of the UK," he said last month.